Nucleic acid sequence encoding FLP recombinase

ABSTRACT

A nucleic acid sequence effectively expressing FLP recombinase in monocot plants, particularly in maize. Stable, transformed maize plants harboring a gene encoding FLP or harboring FRT nucleic acid sequences enable efficient site-directed recombination of nucleic acid sequences in a monocot&#39;s genome.

FIELD OF THE INVENTION

The invention relates to the modification of plant genomes, particularlyto compositions and methods for the excision of nucleotide sequencesthat have been incorporated into the genome.

BACKGROUND OF THE INVENTION

Saccharomyces cerevisiae harbor an autonomously replicating 2T plasmidcontaining the gene FLPy. FLPy encodes the protein, FLP, a site-specificrecombinase acting at defined FLP-recognition targets, FRTs.

Like the cre/lox bacterial system, the yeast FLP/FRT system has beensuggested as providing a useful and efficient method for inserting orexcising genes in a genome of choice. However, in practice, the yeastFLP nucleic acid sequence (FLPy) does not efficiently express activerecombinase in plant cells. For example, in experiments with suspensioncells and callus, FLP-transformed maize cells produced only a very lowfrequency of detectable excision events. See, for example, Lyznik etal., (1993), Nucleic Acids Research 21:969-975 and the Examples below.Furthermore, it has not previously been demonstrated that the FRT/FLPsystem functions in monocot plants, or that FRT or FLP are inheritableand functional in the progeny of monocot plants.

It would therefore be very beneficial to provide an FLP nucleic acidsequence that would efficiently produce an effective FLP recombinase inmonocot plant cells, particularly in maize cells. Such an effective FLPwould permit use of FLP recombinase to selectively and reliably modulategene excision events in a plant cell genome. It would also be veryuseful to provide monocot plants stably transformed with FRT nucleicacid sequences, FLP nucleic acid sequences, or both, capable ofproducing high frequency recombination events, which stably transformedplants pass the FRT/FLP nucleic acid sequences and active recombinase tosubsequent generations.

SUMMARY OF THE INVENTION

Compositions and methods for modification of plant genomes are provided.The compositions comprise FLP recombinase sequences that have beenmodified for enhanced expression in plants. The compositions are usefulfor excision of sequences from the plant genome.

In one embodiment the FLP recombinase is modified for enhancedexpression in maize. This specific nucleic acid sequence, moFLP(Sequence ID. NO: 1) is based on the native, yeast FLP. moFLPefficiently encodes active FLP recombinase (Sequence ID. NO: 2) inmonocot plant cells, particularly in maize cells. Monocot plants,particularly maize plants, are stably transformed with moFLP, andsuccessfully pass the gene onto succeeding generations with fullexpression and activity. Maize plants stably carrying FRT sequences arealso provided, which FRT-containing plants pass these FRT nucleic acidsequences onto succeeding generations. Transgenic monocot plantsexpressing moFLP and containing FRT nucleic acid sequences are capableof producing a high frequency of recombination events (i.e. excision).

Hybrid plants are formed by genetically crossing a plant stablyexpressing moFLP with a plant stably containing FRT sequences. In suchhybrid plants, expression of moFLP permits FLP-mediated recombinationevents at the FRT sequences.

Transformed plants carrying the nucleic acid sequence of at least oneFRT site are modified to excise a nucleic acid sequence from between twoFRT sites, by inducing expression of moFLP in the plant's genome. Suchexpression is induced by transfecting the plant's cells with moFLP, orby inducing expression of moFLP from the plant's genome. Inducedexpression can be via an inducible promoter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a comparison of an optimized sequence of the FLPrecombinase with the native yeast sequence.

DETAILED DESCRIPTION OF THE INVENTION

Novel nucleotide sequences modified for enhanced expression in plantsare provided. The nucleotide sequences encode a FLP recombinase thatprovides functional FLP recombinase activity in the plants.

Expression of the FLP recombinase in a plant cell comprising a targetsequence with flanking FRT sites directs efficient gene excision of thetarget sequence. In this manner, methods are provided for removingunwanted sequences from a plant genome after the sequence has served itsusefulness.

moFLP

The nucleic acid sequence encoding yeast FLP recombinase was modifiedfor enhanced expression in monocots, and particularly for efficientexpression in maize. See, for example the nucleic acid sequence (moFLP)shown in FIG. 1 compared with the yeast FLP nucleic acid sequence (FLPy)(Sequence ID. NO. 3). The 423 amino acid protein sequence of FLP is alsoshown (Sequence ID. NO:2). Recently, a revised sequence for FLPy waspublished, making a single base change at nucleic acid 14 from A to G.This creates a single amino acid change in the FLP protein at amino acid5 from D to G. Compared to the originally published yeast sequence; seeHartley et al. (1980)Nature 286: 860-864.

While the moFLP nucleic acid sequence shown in FIG. 1 includes preferredcodons for expression of amino acids in maize, it is understood that auseful sequence may contain codons occurring in maize with less than thehighest reported maize codon frequencies. For example, those codonshaving a frequency in maize of greater than about 20% need not bealtered to attain an increased efficiency over the yeast sequence. Inthe preferred embodiment, all of the yeast sequence codons having afrequency in maize of less than 20% are changed to codons having afrequency of greater than 20%, using information on maize codon usagesuch as that from Murray et al. (1989) Nucl. Acids Res. 17(2):477-498and from Fennoy et al. (1993) Nucl. Acids Res. 21(23)5294-5300. Apreferred sequence is the modified sequence shown in FIG. 1, moFLP, Seq.ID NO: 1.

In this manner, sequences can be partially modified, substantiallymodified or completely modified. That is, sequences can be constructedcontaining at least 40% modified codons, preferably at least 60%modified codons, more preferably at least 80% modified sequences. Asindicated above, the sequences are modified to contain codons having afrequency in maize of at least 20%.

FRTs

The FRT has been identified as a minimal sequence comprising two 13 basepair repeats, separated by an 8 base spacer, as follows (Sequence IDNo.: 4):

5'-GAAGTTCCTATTC TCTAGAAA!GTATAGGAACTTC3' wherein the nucleotides withinthe brackets indicate the spacer region. The nucleotides in the spacerregion can be replaced with a combination of nucleotides, so long as thetwo 13-base repeats are separated by eight nucleotides. FLP is aconservative, site-specific recombinase, capable of catalyzing inversionof a nucleic acid sequence positioned between two inversely orientedFRTs; recombination between two molecules each containing a FRT site;and excision between FRT sites.

The core region is not symmetrical, and its asymmetry dictates thedirectionality of the reaction. Recombination between inverted FRT sitescauses inversion of a DNA sequence between them, whereas recombinationbetween directly oriented sites leads to excision of the DNA betweenthem. Recombination may also occur at a single FRT site, where atemplate provides matched ends for recombination.

FLP Activity

Assays for FLP recombinase activity are known and generally measure theoverall activity of the enzyme on DNA substrates containing FRT sites.In this manner, a frequency of excision of the target sequence can bedetermined. For example, inversion of a DNA sequence in a circularplasmid containing two inverted FRT sites can be detected as a change inposition of restriction enzyme sites. This assay is described in Vetteret al. (1983) PNAS 80:7284. Alternatively, excision of DNA from a linearmolecule or intermolecular recombination frequency induced by the enzymemay be assayed, as described, for example, in Babineau et al. (1985) JBC260:12313; Meyer-Leon et al. (1987) NA Res 15:6469; and Gronostajski etal. (1985) JBC 260:12328.

Definitions

"Structural gene" is a DNA sequence that is transcribed into messengerRNA (mRNA) which is then translated into a sequence of amino acidscharacteristic of a specific polypeptide.

"Expression" refers to the biosynthesis of a gene product. For example,in the case of a structural gene, expression involves transcription ofthe structural gene into mRNA and the translation of mRNA into one ormore polypeptides.

"Expression vector" is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements, and enhancers.When the gene is under the control of such regulatory elements the geneis said to be operably linked to the regulatory element.

"Promoter" is a DNA sequence that directs the transcription of astructural gene. Typically, a promoter is located in the 5' prime regionof a gene, proximal to the transcription start site of a structuralgene.

"Inducible promoter" is a DNA sequence, that upon exposure to aninducing agent, directs that transcription of the associated structuralgene.

"Constitutive promoter" is a DNA sequence that directs the transcriptionof a structural gene in a relatively steady-state manner, e.g. where therate of transcription is not regulated by an inducing agent.

"Tissue-specific promoter" is a DNA sequence that directs transcriptionof the associated coding region in a specific tissue type(s) such asleaves, root, or meristem.

"Tissue preferred promoter" is a DNA sequence that directs transcriptionof the associated coding region at higher levels in specific tissuetypes such as leaves, root, or meristem.

"Transformed plant cell" A plant cell comprising a DNA region ormodification to DNA introduced as a result of the process oftransformation

"Stably transformed plant" A plant comprising a DNA region ormodification to DNA introduced as a result of the process oftransformation

"Fertile transgenic plant" A plant comprising a DNA region ormodification to DNA introduced as a result of the process oftransformation

"Functional expression of FLP" Production of a FLP site-specificrecombinase protein, through proper transcription of the FLP structuralgene followed by translation into protein, said protein being capable ofbinding to FRT recombination target sites and mediating conservativesite-specific recombination between FRT target sites.

"FLP" A 423 amino acid protein capable of binding to FRT recombinationtarget sites and mediating conservative site-specific recombinationbetween FRT sites.

"FRT site/sites" The recombination target sites recognized by FLPrecombinase protein. The basic configuration consists of a 48 nucleotideDNA sequence consisting of an 8 basepair core and three 13 basepairsymmetry elements where two symmetry elements occur in directorientation on the 5' end of the core sequence and the third elementoccurs in inverted orientation on the 3' end of the core sequence.

"Transient transfection" Introduction of DNA into cells, where theintroduced DNA sequences do not become stably integrated into the genomeof the organism but rather persist as non-integrated molecules whosehalf-life and thus expression are temporally limited based onsusceptibility to endogenous nucleic acid or protein degradation celldivision processes.

Monocot Plant Cell Transformation

Monocot cells, particularly maize cells, can be transformed to includean FRT nucleic acid sequence or a nucleic acid sequence encoding FLP byknown methods for transforming plant cells, as described below.

Gene Transformation Methods

Numerous methods for introducing foreign genes into plants are known andcan be used to insert the FLP gene or FRT nucleic acid sequences into aplant host, including biological and physical plant transformationprotocols. See, for example, Miki et al., (1993), "Procedure forIntroducing Foreign DNA into Plants," In: Methods in Plant MolecularBiology and Biotechnology, Glick and Thompson, eds., CRC Press, Inc.,Boca Raton, pages 67-88. The methods chosen vary with the host plant,and include chemical transfection methods such as calcium phosphate,microorganism-mediated gene transfer such as Agrobacterium (Horsch, etal. (1985) Science 227:1229-31), electroporation, micro-injection, andbiolistic bombardment.

Expression cassettes and vectors and in vitro culture methods for plantcell or tissue transformation and regeneration of plants are known andavailable. See, for example, Gruber, et al., (1993), "Vectors for PlantTransformation" In: Methods in Plant Molecular Biology andBiotechnology, Glick and Thompson, eds., CRC Press, Inc., Boca Raton,pages 89-119.

Agrobacterium-Mediated Transformation

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefacients and A. rhizogenes, respectfully, carry genesresponsible for genetic transformation of plants. See, for example,Kado, (1991), Crit. Rev. Plant Sci. 10:1. Descriptions of theAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provide in Gruber et al., supra; Miki, et al., supra; andMoloney et al. (1989), Plant Cell Reports 8:238.

Direct Gene Transfer

Despite the fact that the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice (Hiei et al.(1994) The Plant Journal 6:271-282) and maize (Ishida et al. (1996)Nature/biotechnology 14:745-750). Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation, where DNA is carried on thesurface of microprojectiles measuring about 1 to 4 μm. The DNA generallycontained in an expression vector expression vector is introduced intoplant tissues with a biolistic device that accelerates themicroprojectiles to speeds of 300 to 600 m/s which is sufficient topenetrate the plant cell walls and membranes. (Sanford et al., (1987)Part. Sci. Technol 5:27; Sanford (1988) Trends Biotech 6:299; Sanford(1990) Physiol. Plant 79:206; Klein et al. (1992) Biotechnology 10:268).

Another method for physical delivery of DNA to plants is sonication oftarget cells as described in Zang et al. (1991) Bio/Technology 9:996.Alternatively, liposome or spheroplast fusions have been used tointroduce expression vectors into plants. See, for example, Deshayes etal. (1985) EMBO J. 4:273 1; and Christou et al. (1987) PNAS USA 84:3962.Direct uptake of DNA into protoplasts using CaCl₂ precipitation,polyvinyl alcohol or poly-L-ornithine have also been reported. See, forexample, Hain et al. (1985) Mol. Gen.Genet. 199:161; and Draper et al.(1982) Plant Cell Physiol. 23:451.

Electroporation of protoplasts and whole cells and tissues has also beendescribed. See, for example, Donn et al. (1990) In: Abstracts of theVIIth Intl. Congress on Plant Cell and Tissue Culture (IAPTC) A2-38,page 53; D'Halluin et al. (1992) Plant Cell 4:1495-1505; and Spencer etal. (1994) Plant Mol. Biol. 24:51-61. Microinjection of DNA into wholeplant cells has also been described as has microinjection intoprotoplasts. See, for example in whole cells, Neuhaus et al. (1987)Theor. Appl. Genet. 75:30-36; and in protoplasts, Crossway et al. (1986)Mol. Gen. Genet. 202:179-185; and Reich et al. (1986) Biotechnology4:1001-1004.

Particle Wounding/Agrobacterium Delivery

Another useful basic transformation protocol involves a combination ofwounding by particle bombardment, followed by use of Agrobacterium forDNA delivery, as described by Bidney et al. (1992) Plant Mol. Biol.18:301-313. Useful plasmids for plant transformation include PHP9762.The binary backbone for PHP9762 is bin 19. See Bevan et al. (1984)Nucleic Acids Research 12:8711-8721.

In general, the intact meristem transformation method involves imbibingseed for 24 hours in the dark, removing the cotyledons and root radical,followed by culturing of the meristem explants. Twenty-four hours later,the primary leaves are removed to expose the apical meristem. Theexplants are placed apical dome side up and bombarded, e.g.) twice withparticles, followed by co-cultivation with Agrobacterium. To start theco-cultivation for intact meristems, Agrobacterium is placed on themeristem. After about a 3-day co-cultivation period the meristems aretransferred to culture medium with cefotaxime (plus kanamycin for theNPTII selection). Selection can also be done using kanamycin.

The split meristem method involves imbibing seed, breaking of thecotyledons to produce a clean fracture at the plane of the embryonicaxis, excising the root tip and then bisecting the explantslongitudinally between the primordial leaves. The two halves are placedcut surface up on the medium then bombarded twice with particles,followed by co-cultivation with Agrobacterium. For split meristems,after bombardment, the meristems are placed in an Agrobacteriumsuspension for 30 minutes. They are then removed from the suspensiononto solid culture medium for three day co-cultivation. After thisperiod, the meristems are transferred to fresh medium with cefotaxime(plus kanamycin for selection).

Transfer by Plant Breeding

Once a single transformed plant has been obtained by the foregoingrecombinant DNA method, e.g., a plant transformed with a desired gene,conventional plant breeding methods can be used to transfer thestructural gene and associated regulatory sequences via crossing andbackcrossing. In general, such plant breeding techniques are used totransfer a desired gene into a specific crop plant. In the instantinvention, such methods include the further steps of: (1) sexuallycrossing an FLP transformed plant with a second FRT transformed plant;(2) recovering reproductive material from the progeny of the cross; and(3) growing FLP/FRT-containing plants from the reproductive material.

Preferred FLP/FRT Systems

The FLP/FRT system is used to advantageously excise a gene from a plantgenome. For example, a marker gene may be inserted into a maize genomealong with a gene of interest. The marker gene is initially useful indemonstrating effective transformation of the plants, but is not desiredin the final product. By inducing expression of FLP, preferably moFLP,transiently or from the plant's genome, the unwanted marker gene isexcised.

In a preferred method of the invention, a first transgenic plant isproduced containing FLP, preferably moFLP, in its genome. A secondtransgenic plant is produced containing FRT nucleic acid sequences,preferably with a gene positioned between at least one pair of FRTs. Bygenetically crossing the first and second transgenic plants, a hybridplant containing both FLP and FRT in the genome is produced. The FLP inthe plant genome is preferably under the control of an induciblepromoter, such as a heat-inducible promoter or an estradiol-responsivepromoter, so that expression of FLP and subsequent excision at the FRTsites is controlled.

Chemical Synthesis of Genes Encoding FLP

The FLP recombinase gene from yeast (Saccharomyces cerevisiae) iscommercially available in plasmid pOG44 from Stratagene Cloning Systems(11011 North Torrey Pines Road, La Jolla, Calif. 92037).

Once the amino acid sequence of FLP is known, a gene encoding FLP can besynthesized. In addition, the nucleotide sequence of a synthetic geneencoding FLP can be optimized for expression in plants by modifying thecodon usage to include plant preferred codons. See, for example, Murrayet al. (1989) NAR 17: 447. Even more specifically, the nucleotidesequence of a synthetic gene encoding FLP can be optimized forexpression in monocotyledonous or dicotyledonous plants. See, forexample, Cambell et al. (1990) Plant Physiol. 92:1. Genes encoding FLPcan be obtained, for example, by synthesizing the genes with mutuallypriming long oligonucleotides. See, for example, Ausubel et al. (eds.),CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pages 8.2.8 to 8.2.13 (WileyInterscience (1990)) "Ausubel"!. Also, see Wosniak et al. (1987) Gene60:115. Moreover, current techniques using the polymerase chain reactionprovide the ability to synthesize genes as large as 1.8 kilobases inlength (Adang et al. (1993) Plant Mol. Biol. 21:1131; Bombat et al.(1993) PCR Methods and Applications 2:266.

Functional fragments of FLP can be identified using a variety oftechniques such as restriction analysis, Southern analysis, primerextension analysis, and DNA sequence analysis. Primer extension analysisor S1 nuclease protection analysis, for example, can be used to localizethe putative start site of transcription of the cloned gene. Ausubel atpages 4.8.1 to 4.8.5; Walmsley et al. "Quantitative and QaulitativeAnalysis of Exogenous Gene Expression by the S1 Nuclease ProtectionAssay," in METHODS IN MOLECULAR BIOLOGY, VOL. 7: GENE TRANSFER ANDEXPRESSION PROTOCOLS, Murray (ed.), pages 271-281 (Humana Press, Inc.1991). Functional fragments of the FLP protein are identified by theirability, upon introduction to cells containing appropriate FRTsubstrates, to catalyze site-specific recombination (for example,excision of an FRT-flanked sequence which upon removal will activate anassayable marker gene.

The general approach of such functional analysis involves subcloning DNAfragments of a genomic clone, cDNA clone or synthesized gene sequenceinto an expression vector, introducing the expression vector into aheterologous host, and screening to detect the product of recombination(i.e. using restriction analysis to verify the product of recombinationat the nucleic acid level, or relying on an assay system forrecombination as described above). Methods for generating fragments of acDNA or genomic clone are well known. Variants of an isolated DNAencoding FLP can be produced by deleting, adding and/or substitutingnucleotides for the isolated nucleotides, for example, the nucleotidesequence of SEQ ID No: 1 (Table 1). Such variants can be obtained, forexample, by oligonucleotide-directed mutagenesis, linker-scanningmutagenesis, mutagenesis using the polymerase chain reaction, and thelike. See, for example, Ausubel, pages 8.0.3-8.5.9. Also, see generally,McPherson (ed.), DIRECTED MUTAGENESIS: A Practical approach, (IRL Press,1991). Thus, the present invention also encompasses DNA moleculescomprising nucleotide sequences that have substantial sequence identitywith SEQ ID NO: 1 and encode FLP. The following terms are used todescribe the sequence relationships between two or more nucleic acids orpolynucleotides: (a) "reference sequence", (b) "comparison window", (c)"sequence identity", (d) "percentage of sequence identity", and (e)"substantial identity".

(a) As used herein, "reference sequence" is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete nucleotide or genesequence.

(b) As used herein, "comparison window" means includes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math.2:482 (1981); by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity methodof Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wisc., USA; the CLUSTAL program is well described byHiggins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS5:151-153 (1989); Corpet, et al., Nucleic Acids Research 16:10881-90(1988); Huang, et al., Computer Applications in the Biosciences 8:155-65(1992), and Person, et al., Methods of Molecular Biology 24:307-331(1994); preferred computer alignment methods also include the BLASTP,BLASTN, and BLASTX algorithms. Altschul, et al., J. Mol. Biol.215:403-410 (1990). Alignment is also often performed by inspection andmanual alignment.

(c) As used herein, "sequence identity" or "identity" in the context oftwo nucleic acid or polypeptide sequences includes reference to theresidues in the two sequences which are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g. charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have "sequence similarity" or "similarity". Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

(d) As used herein, "percentage of sequence identity" means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

(e) (i) The term "substantial identity" of polynucleotide sequencesmeans that a polynucleotide comprises a sequence that has at least 70%sequence identity, preferably at least 80%, more preferably at least 90%and most preferably at least 95%, compared to a reference sequence usingone of the alignment programs described using standard parameters. Oneof skill will recognize that these values can be appropriately adjustedto determine corresponding identity of proteins encoded by twonucleotide sequences by taking into account codon degeneracy, amino acidsequences for these purposes normally means sequence identity of atleast 60%, more preferably at least 70%, 80%, 90%, and most preferablyat least 95%. Polypeptides which are "substantially similar" sharesequences as noted above except that residue positions which are notidentical may differ by conservative amino acid changes.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. to about20° C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typically,stringent wash conditions are those in which the salt concentration isabout 0.02 molar at pH 7 and the temperature is at least about 50, 55,or 60° C. However, nucleic acids which do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides which they encode are substantially identical. This mayoccur, e.g., when a copy of a nucleic acid is created using the maximumcodon degeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is that thepolypeptide which the first nucleic acid encodes is immunologicallycross reactive with the polypeptide encoded by the second nucleic acid.

Construction of Genes and Vectors

The moFLP nucleotide sequence was derived from a back translation of theprotein sequence using maize preferred codons. Sequence analysis wasperformed using the Wisconsin Sequence Analysis Package from GeneticsComputer Group, Madison, Wis. The nucleotide sequence was assembled froma series of synthetic oligonucleotides.

FLP vectors were constructed using standard molecular biologytechniques. See, for example, Sambrook et al. (eds.) MOLECULAR CLONING:a LABORATORY MANUAL, Second Edition, (Cold Spring Harbor LaboratoryPress, cold Spring Harbor, N.Y. 1989). Plasmids are based on pUC18. Thevectors used in these experiments contain combinations of the same basicregulatory elements. The Omega prime (O-) 5-prime sequence is describedby Gallie et al. (1987) Nucl. Acids Res. 15:3257-3273. The selectivemarker gene, bar (Thompson et al. (1987) EMBO J. 6:2519-2523), was usedin conjunction with bailaphos selection to recover transformants. TheCauliflower Mosaic Virus 35S promoter with a duplicated enhancer regionis described by Gardner et al. (1981) Nucl. Acid Res. 9:2871-2888. The79 bp Tobacco Mosaic Virus leader is described by Gallie et al. (1987)Nucl. Acid Res. 15:3257-3273 and was inserted downstream of the promoterfollowed by the first intron of the maize alcohol dehydrogenase geneADH1-S. Described by Dennis et al. (1984) Nucl. Acid Res. 12:3983-3990.The 3- sequence pinII is described by An et al. (1989) Plant Cell 1:115-122.

Promoters

A. Inducible Promoters

An inducible promoter is operably linked to a nucleotide sequenceencoding FLP. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a nucleotide sequence encoding FLP. With an inducible promoter therate of transcription increases in response to an inducing agent.

A variety of inducible promoters can be used in the instant invention.See Ward et al. (1993) Plant Mol. Biol. 22:361-366. Exemplary induciblepromoters include that from the ACE1 system which responds to copper(Mett et al. (1993) PNAS 90:4567-4571); In2 gene from maize whichresponds to benzenesulfonamide herbicide safeners (Hershey et al. (1991)Mol. Gen. Genetics 227:229-237 and Gatz et al. (1994) Mol. Gen. Genetics243:32-38) or Tet repressor from Tn10 (Gatz et al. (1991) Mol. Gen.Genet. 227:229-237. A particularly preferred inducible promoter is apromoter that responds to an inducing agent to which plants do notnormally respond. An exemplary inducible promoter is the induciblepromoter from a steroid hormone gene the transcriptional activity ofwhich is induced by a glucocorticosteroid hormone. Schena et al. (1991)Proc. Natl. Acad. Sci. U.S.A. 88:10421.

The expression vector comprises an inducible promoter operably linked toa nucleotide sequence encoding FLP. The expression vector is introducedinto plant cells and presumptively transformed cells are exposed to aninducer of the inducible promoter. The cells are screened for thepresence of FLP protein by introducing an appropriate FRT-containingsequence, that upon excision, promotes expression of a scorable markersuch as GUS, GFP, luciferase, or anthocyanin production.

B. Tissue-specific or Tissue Preferred Promoters

A tissue-specific promoter is operably linked to a nucleotide sequenceencoding a FLP. Optionally, the tissue-specific promoter is operablylinked to a nucleotide sequence encoding a signal sequence that isoperably linked to a nucleotide sequence encoding FLP. Plantstransformed with a gene encoding FLP operably linked to atissue-specific promoter produce the FLP protein exclusively, orpreferentially, in a specific tissue.

A number of tissue-specific or tissue-preferred promoters can beutilized in the instant invention. Exemplary tissue-specific ortissue-preferred promoters include a root-preferred promoter such asthat from the phaseolin gene (Murai et al. (1983) Science 23:476-482 andSengupta-Gopalan et al. (1985) Proc. Natl. Acad. Sci. USA 82:3320-3324);a leaf-specific and light-induced promoter such as that from cab orrubisco (Simpson et al. (1985) EMBO J. 4(11):2723-2729 and Timko et al.(1985) Nature 318:579-582); an anther-specific promoter such as thatfrom LAT52 (Twell et al. (1989) Mol. Gen. Genet. 217:240-245); apollen-specific promoter such as that from Zm13 (Guerrero et al. (1993)Mol. Gen. Genet. 224:161-168) or a microspore-preferred promoter such asthat from apg (Twell et al. (1993) Sex. Plant Reprod. 6:217-224.

The expression vector comprises a tissue-specific or tissue-preferredpromoter operably linked to a nucleotide sequence encoding FLP. Theexpression vector is introduced into plant cells. The cells are screenedfor the presence of FLP protein by introducing an appropriateFRT-containing sequence, that upon excision, promotes expression of ascorable marker such as GUS, GFP, luciferase, or anthocyanin production.

C. Constitutive Promoters

A constitutive promoter is operably linked to a nucleotide sequenceencoding a FLP or the constitutive promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a nucleotide sequence encoding FLP.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include the promoters fromplant viruses such as the 35S promoter from CaMV (Odell et al. (1985)Nature 313:810-812) and the promoters from such gene as rice actin(McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen etal. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992)Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl.Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730) andmaize H3 histone (Lepetit et al. (1992) Mol. Gen. Genet. 231:276-285andAtanassova et al. (1992) Plant Journal 2(3):291-300).

The ALS promoter, a XbaI/NcoI fragment 5-prime to the Brassica napusALS3 structural gene (or a nucleotide sequence that has substantialsequence similarity to said XbaI/NcoI fragment), represents aparticularly useful constitutive promoter. Co-pending Pioneer Hi-BredInternational U.S. patent application Ser. No. 08/409,297.

The expression vector comprises a constitutive promoter operably linkedto a nucleotide sequence encoding FLP. The expression vector isintroduced into plant cells and presumptively transformed cells arescreened for the presence of FLP protein by introducing an appropriateFRT-containing sequence, that upon excision, promotes expression of ascorable marker such as GUS, GFP, luciferase, or anthocyanin production.

Foreign Protein Genes and Agronomic Genes

According to the present invention, expression of MoFLP to produce FLPrecombinase can be used to modify transgenic sequences that have beenpreviously integrated into the maize genome, through FLP-mediatedexcision of FRT sequences and the intervening DNA sequence. In such amanner, structural genes whose DNA sequence and/or gene-expression arenot desired in the final product can be removed. Thus, marker genes thathave utility in the recovery of transgenic events in culture (or duringplant growth or reproduction) can be removed from a transgenic event,leaving intact, expressing agronomic expression cassettes in the finalproduct. In the process of excising one sequence, a structural gene canalso be moved relative to a promoter to activate the gene (i.e. simplyby moving the structural gene next to the promoter, through the removalof transcriptional impediments such as polyA sequences or stop-codons,or through frame shifts).

Depending on the transformation strategy and the desired final product,many of the genes listed below could be candidates for i) marker genesused for recovery of transgenics during transformation that would laterbe removed from the final commercial product, and also for agronomicallyimportant genes to be expressed in the final product (for example,herbicide genes).

In addition, a marker gene for identifying and selecting transformedcells, tissues, or plants should be included in the transformationconstruct. By marker gene is intended to be either reporter genes orselectable marker genes.

Reporter genes are generally known in the art. The reporter gene usedshould be exogenous and not expressed endogenously. Ideally the reportergene will exhibit low background activity and should not interfere withplant biochemical and physiological activities. The products expressedby the reporter gene should be stable and readily detectable. It isimportant that the reporter gene expression should be able to be assayedby a non-destructive, quantitative, sensitive, easy to perform andinexpensive method.

Examples of suitable reporter genes known in the art can be found in,for example, Jefferson et al. (1991) in Plant Molecular Biology Manual(Gelvin et al., eds.) pp. 1-33, Kluwer Academic Publishers; DeWet et al.(1987) Mol. Cell. Biol. 7:725-737; Goff et al. (1990) EMBO J.9:2517-2522; Kain et al. (1995) BioTechniques 19:650-655; Chiu et al.(1996) Current Biology 6:325-330.

Selectable marker genes for selection of transformed cells or tissuescan include genes that confer antibiotic resistance or resistance toherbicides. Examples of suitable selectable marker genes include, butare not limited to, genes encoding resistance to chloramphenicol(Herrera Estrella et al. (1983) EMBO J. 2:987-992); methotrexate(Herrera Estrella et al. (1983) Nature 303:209-213; Meijer et al. (1991)Plant Mol. Biol. 16:807-820); hygromycin (Waldron et al. (1985) PlantMol. Biol. 5:103-108; Zhijian et al. (1995) Plant Science 108:219-227);streptomycin (Jones et al. (1987) Mol. Gen. Genet. 210:86-91);spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res.5:131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol. 7:171-176);sulfonamide (Guerineau et al. (1990) Plant Mol. Bio. 15:127-136);bromoxynil (Stalker et al. (1988) Science 242:419-423); glyphosate (Shawet al. (1986) Science 233:478-481); phosphinothricin (DeBlock et al.,(1987) EMBO J. 6:2513-2518).

Other genes that could serve utility in the recovery of transgenicevents but might not be required in the final product would include, butare not limited to, such examples as GUS (b-glucoronidase; Jefferson(1987) Plant Mol. Biol. Rep. 5:387), GFP (green flurescence protein;Chalfie et al. (1994) Science 263:802), luciferase (Teeri et al. (1989)EMBO J 8:343) and the maize genes encoding for anthocyanin production(Ludwig et al. (1990) Science 247:449). For certain applications, forexample the commercial production of harvestable protein from transgenicplants as described below, expression of the above genes would bevaluable and thus would remain after excision.

Numerous types of genes fall into the category of potentially valuablegenes that would remain in the final commercial transgenic event afterexcision of various unwanted (or simply unnecessary) transgenicelements. Examples are included below;

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, the selectionand propagation techniques described above yield a plurality oftransgenic plants which are harvested in a conventional manner, and aforeign protein then is extracted from a tissue of interest or fromtotal biomass. Protein extraction from plant biomass can be accomplishedby known methods which are discussed, for example, by Heney et al.(1981) Anal. Biochem. 114: 92-6.

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is maize. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP and PCRanalysis, which identifies the approximate chromosomal location of theintegrated DNA molecule. For exemplary methodologies in this regard, seeGlick and Thompson, METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY269-284 (CRC Press, 1993). Map information concerning chromosomallocation is useful for proprietary protection of a subject transgenicplant. If unauthorized propagation is undertaken and crosses made withother germplasm, the map of the integration region can be compared tosimilar maps for suspect plants, to determine if the latter have acommon parentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR and sequencing, all of which are conventionaltechniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. The genes implicated in this regard include, but are notlimited to, those categorized below including genes that conferresistance to pests or disease and that encode:

plant disease resistance genes (Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al. (1994) Science 266:789(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al. (1993) Science 262:1432 (tomato Pto gene for resistance toPseudomonas syringae pv. tomato encodes a protein kinase); Mindrinos etal. (1994) Cell 78:1089 (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).);

Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon (See, for example, Geiser et al. (1986) Gene48:109, who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection (Rockville, Md.),under ATCC accession Nos. 40098, 67136, 31995 and 31998.);

A lectin (See, for example, the disclosure by Van Damme et al. (1994)Plant Molec. Biol. 24:825, who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.);

A vitamin-binding protein such as avidin (See U.S. patent applicationSer. No. 07/911,864, the contents of which are hereby incorporated byreference. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.);

An enzyme inhibitor, for example, a protease inhibitor or an amylaseinhibitor (See, for example, Abe et al. (1987) J. Biol. Chem. 262:16793(nucleotide sequence of rice cysteine proteinase inhibitor), Huub et aL(1993) Plant Molec. Biol. 21:985 (nucleotide sequence of cDNA encodingtobacco proteinase inhibitor I), and Sumitani et al. (1993) Biosci.Biotech. Biochem. 57:1243 (nucleotide sequence of Streptomycesnitrosporeus α-amylase inhibitor).);

An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof (See, for example, the disclosure byHammock et al. (1990) Nature 344:458, of baculovirus expression ofcloned juvenile hormone esterase, and inactivator of juvenile hormone.);

An insect-specific peptide or neuronpeptide which, upon expression,disrupts the physiology of the affected pest (For example, see thedisclosures of Regan (1994) J. Bio. Chem. 269:9 (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal. (1989) Biochem. Biophys. Res. Comm. 163:1243 (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.); An insect-specific venom produced in nature by a snake, awasp, etc. (For example, see Pang et al. (1992) Gene 116:165, fordisclosure of heterologous expression in plants of a gene coding for ascorpion insectotoxic peptide.);

An enzyme responsible for an hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity;

An enzyme involved in the modification, including the post-translationalmodification, of a biologically active molecule; for example, aglycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease,a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, akinase, a phosphorylase, a polymerase, an elastase, a chitinase and aglucanase, whether natural or synthetic (See PCT application WO 93/02197in the name of Scott et al., which discloses the nucleotide sequence ofa callase gene. DNA molecules which contain chitinase-encoding sequencescan be obtained, for example, from the ATCC under accession Nos. 39637and 67152. See also Kramer et al. (1993) Insect Biochem. Molec. Biol.23:691, who teach the nucleotide sequence of a cDNA encoding tobaccohookworm chitinase, and Kawalleck et al. (1993) Plant Molec. Biol.21:673, who provide the nucleotide sequence of the parsley ubi4-2polyubiquitin gene.);

A molecule that stimulates signal transduction (For example, see thedisclosure by Botella et al. (1994) Plant Molec. Biol. 24:757, ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal. (1994) Plant Physiol. 104:1467, who provide the nucleotide sequenceof a maize calmodulin cDNA clone.);

A hydrophobic moment peptide (See U.S. patent applications Ser. No.08/168,809 (disclosure of peptide derivatives of Tachyplesin whichinhibit fungal plant pathogens) and Ser. No. 08/179,632 (teachessynthetic antimicrobial peptides that confer disease resistance);

A membrane permease, a channel former or a channel blocker. For example,see the disclosure by Jaynes et al. (1993) Plant Sci. 89:43, ofheterologous expression of a cecropin-β lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al. (1990) Ann. Rev.Phytopathol. 28:451. Coat protein-mediated resistance has been conferredupon transformed plants against alfalfa mosaic virus, cucumber mosaicvirus, tobacco streak virus, potato virus X, potato virus Y, tobaccoetch virus, tobacco rattle virus and tobacco mosaic virus. Id.

An insect-specific antibody or an immunotoxin derived therefrom. Thus,an antibody targeted to a critical metabolic function in the insect gutwould inactivate an affected anzyme, killing the insect gut. Cf. Tayloret al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (1994) (enzymatic inactivation in transgenictobacco via production of single-chain antibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al.(1993) Nature 366:469, who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

A developmental-arrestive protein produced in nature by a pathogen or aparasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitate fungalcolonization and plant nutrient release by solubilizing plants cell wallhomo-α-1,4-D-galacturonase. See Lamb et al. (1992) Bio/Technology10:1436. The cloning and characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart et al.(1992) Plant J. 2:367.

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al. (1992) Bio/Technology 10:305, have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease. All of the respectivereferences and the contents thereof are hereby incorporated byreference.

2. Genes That Confer Resistance to a Herbicide, for Example:

A. A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.(1988) EMBO J. 7:241, and Miki et al. (1990) Theor. Appl. Genet. 80:449,respectively.

B. Glyphosate (resistance imparted by mutant EPSP synthase and aroAgenes, respectively) and other phosphono compounds such as glufosinate(PAT and bar genes), and pyridinoxy or phenoxy proprionic acids andcycloshexones (ACCase inhibitor-encoding genes). See, for example, U.S.Pat. No. 4,940,835 to Shah et al., which discloses the nucleotidesequence of a form of EPSP which can confer glyphosate resistance. A DNAmolecule encoding a mutant aroA gene can be obtained under ATCCaccession No. 39256, and the nucleotide sequence of the mutant gene isdisclosed in U.S. Pat. No. 4,769,061 to Comai. European patentapplication No. 0 333 033 to Kumada et al. and U.S. Pat. No. 4,975,374to Goodman et al. disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin-acetyl-transferase gene isprovided in European application No. 0 242 246 to Leemans et al. DeGreef et al. (1989) Bio/Technology 7:61, describe the production oftransgenic plants that express chimeric bar genes coding forphosphinothricin acetyl transferase activity. Exemplary of genesconferring resistance to phenoxy proprionic acids and cycloshexones,such as sethoxydim and haloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3genes described by Marshall et al. (1992) Theor. Appl. Genet. 83:435.

C. A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+genes) and a benzonitrile (nitrilase gene). Przibilla et al.(1991) Plant Cell 3:169, describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC accessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al. (1992) Biochem.J. 285:173.

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearoyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al. (1992) Proc. Natl AcadSci. USA 89:2624.

Decreased phytate content

Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see Van Hartingsveldt et al. (1993) Gene 127:87, for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

A gene could be introduced that reduces phytate content. In maize, this,for example, could be accomplished, by cloning and then re-introducingDNA associated with the single allele which is responsible for maizemutants characterized by low levels of phytic acid. See Raboy et al.(1990) Maydica 35:383.

Modified carbohydrate composition effected, for example, by transformingplants with a gene coding for an enzyme that alters the branchingpattern of starch. See Shiroza et al. (1988) J. Bacteriol. 170:810(nucleotide sequence of Streptococcus mutans fructosyltransferase gene),Steinmetz et al. (1985) Mol. Gen. Genet. 200:220 (nucleotide sequence ofBacillus subtilis levansucrase gene), Pen et al. (1992) Bio/Technology10: 292 (production of transgenic plants that express Bacilluslicheniformis α-amylase), Elliot et al. (1993) Plant Molec. Biol. 21:515(nucleotide sequences of tomato invertase genes), S.o slashed.gaard etal. (1993) J. Biol. Chem. 268:22480 (site-directed mutagenesis of barleyamylase gene), and Fisher et al. (1993) Plant Physiol. 102:1045 (maizeendosperm starch branching enzyme II).

EXAMPLES

The invention may be more fully understood with reference to thefollowing examples, which are not intended to limit the scope of theinvention.

Example I FLP MEDIATED RECOMBINATION IN MAIZE CELL CULTURE

The novel gene of the invention, moFLP (Sequence ID NO:1) was preparedby back translation of the protein sequence using maize preferredcodons. As shown in the Mze₋₋ 90.codtable produced by J. Michael Cherry(cherry@frodo.mgh.harvard.edu). Codons for R and P were furtheroptimized for dicot expression R(CGCyAGG), P(CCGyCCC). Codon L waschanged from CTG to CTC. At base 703, GCC was changed to GCT. Otherchanges put stop codons in all five other reading frames.

Sequence analysis was performed using the Wisconsin Sequence AnalysisPackage from Genetics Computer Group, Madison, Wis. The nucleotidesequence was assembled from a series of synthetic oligonucleotides.Using various vectors and host cells, moFLP was demonstrated asexpressing a functional FLP recombinase in maize plant cells.

A. Recombination Intra-Plasmid and Inter-Plasmid

FLP-mediated recombination within a plasmid (intra-plasmidrecombination) was measured in BMS cell co-transfected with DNA plasmidsexpressing FLP (moFLP or FLPy) and plasmids containing FRT sequences.Two FRT sites were contained in plasmid PHP2729. This plasmid includesthe first FRT site inserted into the first intron from maize Adh1-S;this sequence was followed, in order, by: the coding sequence of themaize gene C1; the potato gene PinII transcription terminator; andplasmid vector DNA sequence. The second FRT site was positioned afterthe latter sequence and in the same orientation as the first FRT site.Following this second FRT site was, in order: the portion of the Adh1gintron sequence that was identical to that portion following the firstFRT site; the coding sequence for luciferase; and the PinIItranscription terminator. In the absence of FLP-mediated recombination,the PHP2729 plasmid expresses the product of the maize gene, C1, whichis one of two classes of transactivators that are both required forexpression of the series of structural genes for the biosyntheticpathway of anthocyanin, a red pigment. In the presence of FLP-mediatedrecombination, expression of C1 is turned off and expression ofluciferase is turned on. Thus, luciferase activity in cells containingthe PHP2729 plasmid indicates recombination between the two FRT sitesdue to FLP recombinase activity.

It is noted that luciferase expression did not have to depend wholly onintra-plasmid recombination, since multiple copies of the plasmid couldhave been introduced into each cell after bombardment. Such multiplecopies could have permitted inter-plasmid recombination between theplasmids resulting in a functional luciferase expression unit. However,luciferase activity does indicate the presence of FLP recombinaseactivity.

FLP-mediated recombination between plasmids (inter-plasmidrecombination) was measured in BMS cells co-transfected with plasmidsPHP2182 and PHP2183. Each of these two plasmids contains a single FRTsite. Upon FLP-mediated recombination between the FRT sites, afunctional luciferase expression unit is created.

Cell Culture

Yeast FRT recombinase sites (FRT-1) were evaluated as substrates for FLPrecombinase activity in BMS cells (genotype Black Mexican Sweet). Thecells were obtained from Dr. David Somers (see Kaeppler, et al., (1990),Plant Cell Reports 9:415-418), University of Minnesota, and grown inliquid suspension medium. The medium included 4.3 g/l MS salts, 0.5 mg/lthiamine-HCl, 150 mg/l L-asparagine, 2 mg/l 2, dichlorophenoxyaceticacid (2,4-D), 20 g/l sucrose, pH 5.8. Cells were incubated at 27° C. ona rotary platform shaker at approximately 100 rpm in the dark. Cellswere subcultured every seven days for maintenance, by transferring 10 mlof suspension into 50 ml of fresh medium.

DNA Particle Bombardment

Cells were resuspended in fresh medium one day prior to bombardment andincubated as described above. On the day of bombardment, cells wereresuspended in osmoticum (MS basal medium, Musashige and Skoog (1962)Physiol. Plant 15:473-497, with 0.25 M sorbitol), at 25 mg cells/ml fora total volume of 60 ml, and incubated as described above for anadditional three hours. One ml of cell-suspension (containing about 25mg cells) was distributed evenly into petri dishes (60 mm diameter×20 mmheight) containing two paper filter disks (Baxter S/P #363, 5.5 cmdiameter), prewetted with one ml osmoticum, onto the top filter. Thecells on the filter were used as the bombardment target.

For particle bombardment, plasmid DNA (described below) was precipitatedonto 1.8 μm tungsten particles using standard CaCl₂ -spermidinechemistry (see, for example, Klein et al. (1987) Nature 327:70-73). Eachplate was bombarded once, using a DuPont Helium Gun (Lowe et al. (1995)Bio/Technol 13:677-682). After bombardment, an additional 0.5 ml ofosmoticum was added to each plate. All plates were incubated in the darkat 27° C. for two days, after which crude extracts were prepared andassayed for luciferase activity and total cell protein.

The plasmids used in this study were designed to demonstrate functionalFLP recombinase activity and FRT recombination in maize cells throughtransient transgene expression, driving the reaction entirely withplasmid-borne templates. Plasmids used in these studies included thoseshown below in Tables I-A-1 and I-A-2:

For features of plasmid DNAs for expressing FLP protein:

                  TABLE I-A-1    ______________________________________                     Other 5'    Structural                                         3'    PHP    Promoter  Elements    Gene    Elements    ______________________________________    4894   Enhanced  Omega'/Adh-1                                 MoFLP   PinII           CaMV35S   intron              terminator    5095   Ubi-1     Ubi-1 exon 1                                 FLPy    PinII                     Ubi-1 intron 1      terminator    5096   Ubi-1     Ubi-1 exon1 MoFLP   PinII                     Ubi-1 intron1       terminator    ______________________________________

For features of plasmid DNAs containing FRT(s):

                                      TABLE I-A-2    __________________________________________________________________________    PHP    Promoter                 Other 5' Elements                         Structural Gene                                3' Elements                                      Note    __________________________________________________________________________    2175   Enhanced                 omega'/Adh1                         Luciferase                                PinII Positive control for           CaMV35S                 intron, with   Terminator                                      PHP2729 and for                 FRT site             combination of                 inserted into        PHP2182 + PHP2183                 Adh1 intron    2182   No promoter                 FRT/(3' Luciferase                                PinII Does not express                 portion of     Terminator                                      Luciferase; if correctly                 Adh1 intron)         recombined with                                      PHP2183, reconstitutes                                      functional expression                                      unit    2183   Enhanced                 omega'/Adh1                         GUS    PinII See Comment for           CaMV35S                 intron, with   terminator                                      PHP2182 above                 FRT site                 inserted into                 Adh1 intron    2729 (first           Enhanced                 omega'/Adh1                         Cl     PinII In absence of FLP    group of           CaMV35s                 intron, with   Terminator                                      mediated recombination,    elements)    FRT site             expresses Cl but not                 inserted into        Luciferase; with FLP                 Adh1 intron          protein mediated                                      recombination between                                      FRT sites, expresses                                      Luciferase not Cl    2729   No promoter                 FRT/(3' Luciferase                                PinII same orientation as    (second group                 portion of     Terminator                                      first group,    of elements) Adh1 intron)         downstream (3') of first                                      group, and separated                                      by intervening vector                                      sequence.    __________________________________________________________________________

Cells were bombarded with particular combinations of plasmids to achievethe experimental designs shown below in Table I-A-3.

For an experimental design:

                                      TABLE I-A-3    __________________________________________________________________________    Treat-        PHP2729             PHP5095                  PHP5096                       PHP2182                            PHP2183                                 PHP4894                                      PHP2175    ment #        FRT  FLPy MoFLP    F   RT---             MoFLP                  FRT  Note    __________________________________________________________________________    1   5 μg             1 μg                       assay for    2   5 μg   1 μg                  intra-plasmid    3   5 μg   0.2 μg                recombination    4   5 μg   0.04 μg    5   5 μg    6                  3 μg                            3 μg        assay for    7                  3 μg                            3 μg                                 0.04 μg                                           inter-plasmid    8                  3 μg                            3 μg                                 0.2 μg recombination    9                  3 μg                            3 μg                                 1 μg   control    10                                0.01 μg                                           positive    11                                0.1 μg                                           controls for    12                                1.0 μg                                           treatments    13                                5.0 μg                                           #1-9 above    __________________________________________________________________________

Luciferase Assay

Two days after particle bombardment, cells were examined for C1 activity(red pigment) and assayed for luciferase activity. For the luciferaseassay, cells were scraped from the filter paper of each plate,transferred to a 1.5 mm Kontes microfuge tube and lysed by grinding in200 μl lysis buffer (40 mM sodium phosphate, pH 6.8, 1 mM EDTA, 50 mMβ-mercaptoethenol) while keeping cool on ice. All samples werecentrifuged at 4000×g, 15 minutes at 4° C. and kept on ice. A volume of20 μl of each extract was added to 200 μl of room temperature luciferaseassay buffer (25 mM tricine, pH 7.8, 15 mM MgCl₂, 5 mM ATP, 500 μg/mlBSA) in a cuvette. The cuvette was immediately placed into an AnalyticalLuminescence Laboratories Monolight 2010 Luminometer with automaticinjection of 100 μl of 0.5 mM D-luciferin (potassium salt) to start theluminescence action. The device collected relative light units (R.L.U.)for a duration of 10 seconds.

Duplicate reactions for each of the samples described above were set upin separate wells of flat-bottom transparent microtitre plates, byadding 159 microliters of water, one microliter of crude extract and,lastly, 40 microliters BioRad Laboratories Bradford Dye Concentrate, andthen mixing by repeated pipetting. After 5 to 45 minutes, the opticaldensity at 595 nm was measured with a micro plate spectrophotometer. Theamount of protein present was estimated using a standard curve of BSAprotein.

Recombination Between Plasmids Inter-plasmid and Recombination

moFLP produced active FLP protein, promoting intra-plasmid andinter-plasmid recombination. As shown in data table I-A-4 below(treatments 6-9), plasmid PHP4894 containing moFLP expressed sufficientFLP recombinase to promote the necessary inter-plasmid recombinationrequired for reconstitution of a functional expression unit forluciferase from plasmids PHP2182 and PHP2183.

Also shown below in data table I-A-4 (treatments 1-5), both FLPy andmoFLP produce active LP protein, promoting intra-plasmid recombination.Recombinase activity expressed in luciferase units appeared to be higherfrom moFLP (treatments 2-4), as compared with FLPy (treatment 1).

                  TABLE I-A-4    ______________________________________            Luciferase Activity (R.L.U./μg total protein)                                   Mean %**                          Standard templates    Treatment #              Mean        Deviation                                   recombined    ______________________________________    1         667         187        16%    2         776         216        20%    3         1301        254        26%    4         1026        107        22%    5         4.9         4.2       2.2%    6         3.5         0.8       2.7%    7         93.7        51.9     12.7%    8         107.4       55.9     13.3%    9         182.8       24       17.3%    10        0.7         1.0      --    11        3.5         3.2      --    12        759         481      --    13        14916       9665     --    ______________________________________     *n = 3     **based on standard curve of dose response series of the positive control     PHP2175

B. Confirmation of FLP-Mediated Recombination

The FLP-mediated intra-plasmid and inter-plasmid recombination studiesdescribed above for Example IA were repeated, and as shown in TableI-B-1, confirmed the increase FLP recombinase activity of moFLP(treatments 2-4) over FLPy (treatment 1).

                  TABLE I-B-1    ______________________________________            Luciferase Activity (R.L.U./μg total protein)*                                   Mean %              Mean        Standard templates    Treatment #              Luciferase  Deviation                                   recombined,    ______________________________________    1         290.2       189.9    31.4%    2         399.9       228.2    47.0%    3         348.6       48.1     39.2%    4         463         143      49.7%    5         22.0**      22.1**     1.0%**    6         4.0         1.1       0.2%    7         31.3        17.0     0.82%    8         23.9        7.4      0.82%    9         18.5        5.6      0.75%    10        2.6         10.5    11        49.6        34.3    12        228.5       33.8    13        1387.8      288.7    14        -0.6        0.8    ______________________________________     *n = 3     **n = 2

C. moFLP is More Active at Lower Doses

Transient expression and dose responsive FLP recombinase activity ofFLPy versus moFLP was assessed using the FRT and FLP plasmids describedabove and using the methods described for Example IA. Specifically,varying amounts of FRT and FLP-expresing plasmids were used, as shownbelow in experimental design table I-C-1. To mantain a constant level of35S promoter-containing plasmids, control plamid PHP611 was added asrequired. This plasmid contains the BAR structural gene driven by theEnhanced CaMV35S promoter.

                                      TABLE I-C-1    __________________________________________________________________________    Table Describing Combination of Mass and Type of Input Plasmid DNAs          PHP2729               PHP5095                    PHP5096                         PHP2182                              PHP2183                                   PHP4894                                        PHP2175    Treatment #          FRT  FLPy MoFLP                         FRT  FRT  Control                                        Control    __________________________________________________________________________    1     5 μg               1 μg    2     5 μg               0.02 μg    3     5 μg               0.004 μg    4     5 μg               0.0008 μg    5     5 μg   0.1 μg    6     5 μg   0.02 μg    7     5 μg   0.004 μg    8     5 μg   0.0008 μg    9          0.02 μg                         5 μg                              5 μg    10              0.02 μg                         5 μg                              5 μg    11                             0.01 μg                                        10 μg    12                             0.1 μg                                        9.9 μg    13                             1 μg                                        9 μg    14                             10 μg    15    5 μg    16                   5 μg                              5 μg    __________________________________________________________________________

Cells were cultured and prepared for bombardment, as described above forExample IA. Appropriate amounts of plasmid DNA, as shown in Table I-C-1,were used to coat particles and bombard the cells as described above forExample IA. Assay of luciferase activity and total protein andcalculation and expression of the data were performed as described abovefor Example IA. As shown below in data table I-C-2, the moFLP DNA codingsequence in plasmid PHP5096 (treatments 5-8, 10) conferred higher ratesof expression by means of increased efficiency of translation, ascompared to the FLPy sequence in PHP5095 (treatments 1-4, 9). This trendis suggested by recombination events using both types of plasmidtemplate series, PHP2729 for intra-plasmid recombination (treatments1-8) and plasmids PHP2182 and PHP2183 for inter-plasmid recombination(treatments 9-10).

                  TABLE I-C-2    ______________________________________                Luciferase Activity (R.L.U./μg total protein)    Treatment #   Mean       Standard Deviation    ______________________________________    FLPy    1     1.0     56.0     22.3            2     0.02    12.6     4.1            3     0.004   14.2     12.5            4     0.0008  13.5     2.3    moFLP   5     0.1     190.1    15.3            6     0.02    203.8    83.8            7     0.004   105.7    27.2            8     0.0008  15.6     5.5    FLPy    9     0.02    30.1     5.4    moFLP   10    0.02    88.0     24.6    CTR     11            0.3      0.1    CTR     12            11.4     3.7    CTR     13            1058     347    CTR     14            3249     603    CTR     15            10.0     7.0    CTR     16            25.3     4.2    ______________________________________

The data tables shown above demonstrate the gene of the invention, moFLPproduced more recombinase activity in maize cells than did FLPy for bothintra-plasmid recombination and inter-plasmid recombination.

D. Dose Response FLP

Reinforcing the results in Example I-C, moFLP produces higherrecombination frequency at lower plasmid levels, compared to FLPy (whensubstrates are co-delivered with the FLP expression plasmid). The FLPdose response study of Example I-C comparing FLPy and moFLP was repeatedand expanded using higher amounts of FLPy, as shown below in theexperimental design table, Table I-D-1.

                                      TABLE I-D-1    __________________________________________________________________________          PHP2729               PHP5095                    PHP5096                         PHP2182                              PHP2183                                   PHP4894                                        PHP2175    Treatment #          FRT  FLPy moFLP                         FRT  FRT  Control                                        Control    __________________________________________________________________________    1     5 μg               2.5 μg    2     5 μg               0.5 μg    3     5 μg               0.1 μg    4     5 μg               0.02 μg    5     5 μg   0.1 μg    6     5 μg   0.02 μg    7     5 μg   0.004 μg    8     5 μg   0.0008 μg    9          0.02 μg                         5 μg                              5 μg    10              0.02 μg                         5 μg                              5 μg    11                             5.0 μg                                        5.0 μg    12                             1.0 μg                                        9.0 μg    13                             0.1 μg                                        9.9 μg    14                             0.01 μg                                        10.0 μg    15    5 μg    16                   5 μg                              5 μg    __________________________________________________________________________

Cells were incubated and bombarded with plasmid DNA, as described abovefor Example 1A. Luciferase activity and total protein was assayed andcalculated, as described above. As shown below in data table I-D-2,trends observed in the earlier experiments for recombination ofFRT-containing plasmid DNA's co-delivered with plasmids coding for FLPexpression were continued. Use of the plasmid containing moFLP producedmore recombinase activity than FLPy for intra-/inter-plasmidrecombination using PHP2729 as substrate (treatments 1-4=FLPy;treatments 5-8=moFLP). Plasmids containing moFLP of the invention alsodemonstrated expression of higher recombinase activity mediatinginter-plasmid recombination than that expressed from FLPy plasmids whenPHP2182 and PHP2 183 were co-delivered to cells as substrates(treatments 9 and 10). The efficiency of expression was based on linearportions of dose response curves for PHP5095 and PHP5096.

                  TABLE I-D-2    ______________________________________                  Luciferase Activity                  (R.L.U./μg total protein)*                    Mean               Mean %                    Luciferase                             Standard  Templates    Treatment #     Activity Deviation recombined*    ______________________________________    FLPy   1       2.5 μg                            5756   3240    23.50%            2**    0.5 μg                            567    265     9.93%           3       0.1 μg                            146.2  38.2    1.05%           4       0.02 μg                            132.6  30.9    0.92%    moFLP  5       0.1 μg                            2372   766     23.50%           6       0.02 μg                            1547   403     5.20%           7       0.004 μg                            1735   377     9.20%           8       0.008 μg                            362    82      2.60%    FLPy   9       0.02 μg                            69.5   35.7    0.28%    moFLP  10      0.02 μg                            443.4  174.7    1.6%    Control           11               29480  4402    Control           12               6727   2986    Control           13               451.4  26.8    Control           14               35.9   3.8    Control           15               21.5   2.0    Control           16               26.9   8.6    ______________________________________     *n = 3 based on standard curve of dose response series of the positive     control, PHP2175     **n = 2

E. Evaluation of FRT and FLP Constructs in an Embrvogenic Callus

In Embryogenic maize callus, the efficiency of moFLP was about 35 foldthat of FLPy. The transient expression assays described above forassessment of the efficiency of plasmids expressing moFLP and FLPy wererepeated using maize embryogenic callus obtained from #3-44-6E1, similarto the culture described in Ludwig, et al., (1990), Science 247:449-50.#3-44-6E1 is a cross between female parent (wx5-9, w23) and male parent(HD2233 A63). To prepare the cell suspension, immature embryos(approximately 1.5 mm in length) were harvested from ears approximately10-12 days after pollination. The embryos were placed axis down(scutellum up) on culture initiation medium. For example, a typicalculture initiation medium contains N6 salts, Erikkson's vitamins, 0.69g/l proline, 2 mg/l 2,4-D and 3% sucrose. Friable callus initiates fromthe scutellum and is subcultured periodically onto fresh medium. Friablecallus is then placed into liquid medium on a rotary shaker, usingwell-established methods for subcuturing and maintenance of theresulting suspension culture. For an example of standard media andmethods for initiation, maintenance of friable, type II callus andsuspension cultures see Sellmer et al., (1994), In "The Maize Handbook",M. Freeling and V. Walbot, eds. Springer-Verlag, New York, pp 671-684.

The cell suspension was routinely maintained in liquid medium (4.3 g MSsalts/l, 0.1 g myo-inositol/l, 0.5 mg/l/nicotenic acid, 0.02 mg/lthiamine-HCl, 0.5 mg/l pyrodoxine-HCl, 2 mg/l L-lysine, 2 mg/l 2, 4-D,30 g sucrose/l, final pH 5.6) in the dark at 27° C. on a rotary platformshaker at about 100 rpm, and at 1-3 gram cell weight/60 ml medium.

Cells were resuspended in fresh medium on the day prior to bombardment.Two hours prior to bombardment, cells were resuspended at 1.5 gram wetweight per 60 ml osmoticum and incubated. After the incubation, one mlaliquots of cell suspension were pipetted onto pre-wetted paper filterdiscs in petri dishes, as described above for Example I-A.

Particle bombardment was carried out using the FRT and FLP-containingplasmids and methods described above for Examples IA-ID. Control plasmidPHP610 (Enhanced CaMV35s::AdhI intron::bar::pinII) expressing the bargene was used as an indicator of bombardment efficiency. Afterbombardment, additional osmoticum (0.25-0.5 ml) was added to each plateand the plates then incubated in the dark at 27° C. for one day. Thecells were then harvested, crude extracts prepared, and samples assayedfor luciferase activity and total cell protein as described above. Theexperimental design is shown in table I-E-1 below:

                  TABLE I-E-1    ______________________________________            PHP2729  PHP610   PHP5095                                     PHP5096                                            PHP2175    Treatment #            FRT      Control  FLPy   moFLP  FRT    ______________________________________    1       5 μg  5 μg    2       5 μg  2.5 μg                              0.02 μg    3       5 μg  2.4 μg                              0.1 μg    4       5 μg  2.0 μg                              0.5 μg    5       5 μg           2.5 μg    6       5 μg  2.5 μg       0.0008 μg    7       5 μg  2.5 μg       0.004 μg    8       5 μg  2.5 μg       0.02 μg    9       5 μg  2.4 μg       0.1 μg    10               10 μg               0.01 μg    11               9.9 μg              0.1 μg    12               9.0 μg              1.0 μg    13               5.0 μg              5.0 μg    ______________________________________

The cells were bombarded, incubated, and tested for luciferase activityand total protein, as described above. The data, shown below in TableI-E-2 indicates the same trends observed using BMS cells. The plasmidexpressing maize-optimized moFLP (treatments 6-9) was more efficient inexpressed FLP recombinase activity than the plasmid expressing FLPy(treatments 2-5). The efficiency of moFLP was about 35 fold that of FLPyin this experiment.

                  TABLE I-E-2    ______________________________________                Luciferase Activity                (R.L.U./μg total protein)*    Treatment #   Mean    Standard Deviation    ______________________________________    1 Control     5.4     2.9    2 FLPy        114.3   52.9    3             245.3   105.5    4             808.5   223.4    5             1248.9  322.9    6 moFLP       113.3   115.8    7             315.7   106.3    8             446.2   297.9    9             295.2   231.3    10 Control    2.8     1.6    11            23.5    10.6    12            194     275.4    13            742.9   463.5    ______________________________________     *n = 3

F. Ubi and CaMV35S Promoters for Expression of FLP

Ubi-driven moFLP expression produced higher levels of FLP-mediatedexcision than the Enhanced CaMV35S promoter, and the addition of FRT tothe FLP coding sequence did not abolish activity of the FLP. Theexpression of moFLP under the control of the Ubiquetin (Ubi) and the 35SCauliflower Mosaic Virus (CaMV 35S) promoters was compared. In addition,expression of moFLP having an FRT site at the N-terminus of its codingsequence was evaluated. The specific plasmids used are described belowin Table I-F-1.

For features of plasmid DNAs for expressing FLP protein:

                  TABLE I-F-1    ______________________________________    Plas-                    Struc-                                   3'    mid           Other 5'   tural Ele-    PHP  Promoter Elements   Gene  ments                                        Note    ______________________________________    5849 Enhanced omega'     moFLP PinII                                        If FRT does not         CaMV     Adh1 intron           interfere with FLP         35S      I; FRT                expression, then,                                        when moFLP is                                        excised another                                        structural gene will                                        be moved adjacent                                        to the CaMV pro-                                        moter, activating                                        this second gene    3456 Enhanced omega'; Adh1                             GUS   PinII                                        GUS(-) until FLP-         CaMV     intron I; ATG         mediated excision of         35S      codon; FRT;           intervening 1.8 KB                  ˜1.8 KB Adh1    AdhI sequence                  intervening                  sequence; FRT    3457 Enhanced omega'; 5' GUS   PinII                                        GUS(+) the ex-         CaMV     portion of            pected product after         35S      Adh1 Intron I;        excision in                  ATG codon;            PHP3456                  FRT    5401 Ubi-1    Ubi-1 exon 1 +                             GUS   PinII                                        GUS(-) until FLP-                  intron 1; ATG         mediated excision of                  codon; FRT;           intervening 1.8 KB                  ˜1.8 KBAdhI     AdhI sequence                  sequence; FRT    ______________________________________

The experimental design is shown below in Table I-F-2:

                                      TABLE I-F-2    __________________________________________________________________________          PHP5096               PHP3456                     PHP5849                           PHP5401                                 PHP3457    Treatment          Ubi- CaMV  CaMV  Ubi-  GUS  PHP610    #     moFLP               FRT   FRT/moFLP                           FRT   Control                                      Control    __________________________________________________________________________    2          10 μg    3          10 μg                     0.004 μg    4          10 μg                     0.02 μg    5          10 μg                     0.1 μg    6          10 μg                     0.5 μg    7     0.0008 μg               10 μg    8     0.004 μg               10 μg    9     0.02 μg               10 μg    10    0.1 μg               10 μg    11    0.0008 μg     10 μg    12    0.004 μg      10 μg    13    0.02 μg       10 μg    14    0.1 μg        10 μg    19                           0.01 μg                                      10 μg    20                           0.1 μg                                      9.9 μg    21                           1.0 μg                                      9.0 μg    22                           10 μg    24                     10 μg    __________________________________________________________________________

Culture conditions, DNA particle bombardment and assays were performedas described above for Examples IA-IE. GUS fluorescence assay wasperformed as described in Rao and Flynn (1990) Biotechnique 8:38-40.

As shown below in Data Table 1-F-3, FRT-containing plasmids were silentfor transient expression of reporter GUS in the absence of co-deliveredFLP expression vector (treatments 1-2), but were activated to expressionlevels 100-1000 fold over that of background in the presence ofco-delivered FLP. Addition of FRT to the FLP coding sequence in PHP5849did not abolish activity of the FLP (treatments 3-6). Use of the Ubipromoter resulted in greater FLP-mediated excision activity thanpreviously seen with the CaMV35S promoter.

                  TABLE I-F-3    ______________________________________                  GUS Fluorescent Units/                  μg total protein    Treatment #             Standard    (n = 3)         Mean    Deviation    ______________________________________    1               1.46    0.29    2               1.90    0.70    3               6.43    4.47    4               49.9    6.0    5               254     71.1    6               279     27.7    7               72.3    12.2    8               216     79.6    9               391     95.0    10              465     186    11              572     168    12              888     264    13              2216    268    14              4622    1175    15              2.21    7.77    16              15.12   17.63    17              49.2    14.8    18              564     144    19              7.65    7.23    ______________________________________

Example II FLP MEDIATTED EXCISION IN MAIZE CELLS

Transient expression of moFLP will excise an FRT-delimited sequence fromthe genome of maize cells. Transgenic maize cell lines containing FRTsites integrated into the genome were produced as described below. Thesecells were then used to evaluate FLP-mediated excidion of FRT-flankedintegrated DNA sequences.

A. Intergration of FRT Sites in Maize Genome

BMS cells were obtained from Dr. Somers, as described above. Cells wereincubated and maintained as describie above in Example I, for culture ofBMS cells in medium 237.

To produce transformed cells containing integrated FRT sequences, theFRT-containing plasmid PHP5401 (described above for Example I-F, TableI-F-1) and the control, ALS #2-containing plasmid PHP2545 containing themaize ALS#2 structural gene conferring GLEAN® resistance driven by theEnhanced CaMV35S promoter with the Omega' element, the Adh1 gintron Iand the nos 3' element) were used. Cells transformed with PHP5401express GUS upon FLP-mediated excision of a 1.8 kb Adh-1 sequence frombetween the plasmid's FRT sequences. Cells transformed with the controlPHP2545 express the maize gene ALS#2, which confers resistance to GLEAN®(Dupont, Inc.). GLEAN® resistance was used to screen for transformedcells.

Cells co-transformed with the FRT-containing plasmid PHP5601 and theALS#2-containing plasmid PHP2545 were initially screened for GLEAN®resistance. For each plate of cells after bombardment, the top filterwith cells was transferred to a plate of #306 medium (4.3 g MS salts/L,0.1 g myo-inositol/L, 2 mg/l L-glycine, 1 mg/L 2,4-D, 30 g/L sucrose, 3g/l Gelrite, final pH=5.6). The cells were incubated at 27° C. in theabsence of light, for 3 days. The cells were then resuspended in 4 ml#237 medium and pipetted into 4 plates of #306 N medium (#306 mediumplus 20 ppb GLEAN®) at 1 ml cell suspension/plate. Independenttransformants were subcultured to separate plates of #306N as theyappeared over the next 6-8 weeks. GLEAN®-resistant transformants wereobtained and used to assay for FRT transformants using transientexpression of FLP as described more fully below.

B. Screening for FRT Sequences Using FLP

The GLEAN®-resistant transformants produced as described above, werescreened by transient FLP expression, for co-transformants carryingintegrated FRT-containing transgenic plasmid DNA sequences. In summary,transformed cells were bombarded with DNA particles containing anFLP-expressing plasmid (PHP5096, as described for Example I, TableI-A-1). The experimental design is shown below in Table II-B-1. Thepositive control FRT-containing PHP5401 is described in Example I, TableI-F-1. The negative Control PHP687 containing anthocyanin genes C1 and R(Ludwig, et al., (1990), Science 247:449-451). This plasmid, PHP687,contained two structural genes, R and C1, each driven by EnhancedCaMV35S, with the Omega' element and the Adh1 intron I, and followed bythe 3' pinII sequence. The bombardment protocol was that described abovefor Example I. Cells were then screened for GUS expression, indicativeof FLP-mediated excision events.

                                      TABLE II-B-1    __________________________________________________________________________                  Total number    Treat-        of plates shot,                         PHP5096                              PHP687                                    PHP5401    ment #        Cells     5 trfs/plate                         (moFLP)                              (CI Control)                                    (FRT)                                         Note    __________________________________________________________________________    Resistant ®                  14     1 μg                              0.5 μg        transformants        treatment #1 (N = 66)    2   BMS Control Cells                  3      1 μg                              0.5 μg  Negative control-                                         expect Cl and no                                         GUS+    3   BMS Control                  3      1 μg                              0.5 μg                                    8 μg                                         Positive control for                                         FLP expression-                                         +GUS assay, +Cl    __________________________________________________________________________

Following bombardment, fresh medium was added to samples, which wereincubated for two days at 27° C. in the dark. Cells were assayed forexpression of C1 (red cells) versus GUS (blue cells). In the GUS assay,after another day of incubation, the bottom filters on each petri dishwere replaced with dry ones, and 1 ml X-Gluc stain (50 mm NaPO₄, pH 7.0,0.1% X-Gluc, 0.1% Triton X100 0.1% sarkosyl, 1.25% DMSO) was added toeach plate. Samples were then examined for blue-staining foci, (GUS+)indicative of FLP expression and activity at FRT sites in BMS-FRT cells.

The results are shown below in Table II-B-2. At least two events werepositive for carrying transgenic FRT DNA sequences accessible to FLPexpressed transiently.

                  TABLE II-B-2    ______________________________________            Sam-            ple    Red cell counts                              Events producing    Treat-  size   (transfection                              blue cells    ment #  (n)    efficiency - C1)                              (GUS+)    Note    ______________________________________    1       66     Ranged from                              Event # "1A-14"                                        Nearly all foci    GLEAN ®    <10 to >400                               1 blue cell!                                        appeared to be    resistant      per trans-  >100 red cells!;                                        restricted to    BMS-FRT        formant    Event # "1B-12"                                        one cell; not                               131 foci!  ˜50                                        as intense as                              red cells!                                        observed in                                        treatment                                        #3 below.    2       3      >100       None    Negative    Control    3       3      >100       >100      Foci hetero-    Positive                            geneous in    Control                             staining                                        intensity (many                                        very intense,                                        wide, as usu-                                        ally observed                                        in past)    ______________________________________

C. FLP Activation of Genomic FRT Sites

Transcormed maize cells carrying integrated FRT sites prepared forExample II-A (transformants -1 and -2) were maintained on #306N medium.To confirm FLP-mediated activation of integrated FRT sites inFRT-transformed cells, the FLP-transient expression assay of ExampleII-B was repeated, using a control plasmid to monitor DNA delivery. Theexperimental design is shown below in Table II-C-1. PHP3528 is anon-FLP, non-GUS, BAR-expressing control plasmid. PHP687 is a controlplasmid expressing R and C1 proteins, and is co-delivered to assess theefficiency of DNA delivery.

                  TABLE II-C-1    ______________________________________                          PHP   PHP                   PHP    687   3528  PHP    Treat-         5096   (con- (con- 5401    ment #          Cell line                   (FLP)  trol) trol) (FRT) Note    ______________________________________    1     Trans-   1 μg                          0.5 μg                                --    --          formant-1    2     Trans-   1 μg                          0.5 μg                                --    --          formant-2    3     BMS-     1 μg                          0.5 μg                                --    --    Negative con-          control                           trol for GUS                                            expression                                            from input                                            DNAs    4     Trans-   --     0.5 μg                                1 μg                                      --          formant-1    5     Trans-   --     0.5 μg                                1 μg                                      --    Controls for          formant-2                         GUS back-                                            ground expres-                                            sion    6     BMS-     --     0.5 μg                                1 μg                                      --          control    7     BMS-     1 μg                          0.5 μg                                --    8.5 μg                                            Positive control          control                           for FLP and                                            GUS expres-                                            sion    8     BMS-     --     --    --    --    Un-shot control          control    ______________________________________

As shown below in Data Table II-C-2, BMS cell lines were stablytransformed with transgenic FRT-DNA sequences. These stabletransformants activated GUS expression after introduction of moFLP bytransient expression, confirming the results shown above in TableII-B-2.

                                      TABLE II-C-2    __________________________________________________________________________                 Input DNAs for GUS cytochemical assay and for red cell                 counts (C1)                                   PHP5096                                   +                 PHP5096  PHP3528  PHP5401                 +        +        +                 PHP687   PHP687   PHP687      Non-shot                 (moFLP)  (control)                                   (FLP/FRT control)                                               cells    Cell line           Marker                 A  B  c  A  b  c  a   b   c   a b c    __________________________________________________________________________    Transformant-1           # GUS foci:                 1  0  0  0  0  0              0           # Red cells:                 +++                    +++                       +++                          +++                             +++                                +++    Transformant-2           # GUS foci:                 92 121                       133                          0  0  0              0           # Red cells:                 +++                    +++                       +++                          +++                             ++ +++    BMS    # GUS foci:                 0  0  0  0  0  0  >1000                                       >1000                                           >1000                                               0 0 0           # Red cells:                 +++                    +++                       +++                          +++                             +++                                +++                                   +++ +++ +++ 0 0 0    __________________________________________________________________________

These data demonstrate that transiently expressed moFLP exertsrecombinase activity on genomic FRT-containing DNA to catalyzesite-specific excision of DNA intervening sequences between directlyrepeated FRT sites.

D. moFLP Versus FLPy Mediated Genomic Excision

The moFLP gene produces higher levels of site-specific excision in maizecells than FLPy. The FRT-containing BMS transformants produced inExample IIA were transfected with varied doses of FLP-expressing vectorsfor site specific excision of genomic transgenic sequences using themethods described above for Example II-C. Plasmids expressing FLP,PHP5095 (FLPy) and PHP5096 (moFLP) are described above for Example I-A(see Table I-A-1). The FRT/moFLP-containing PHP5849 is described abovefor Example I-F (see Table I-F-1). Control plasmids used were PHP3703(non-FLP negative control; with UBI promoter and intron, wheat germagglutinin structural gene and pinII 3' end), PHP3953 (positive control;with UBI promoter and intron, GUS structural gene and pinII 3' end), andpUC18 with no plant expression cassette used as carrier DNA to keep thetotal DNA load constant). Cell culture methods, DNA bombardment methods,and assay methods were those described above for Example I. Theexperimental design is shown below in Table II-D-20 1.

                                      TABLE II-D-1    __________________________________________________________________________              PHP  PHP  PHP 5849    Treat-        Cell  5095 5096 (moFLP/                             PHP PHP    ment #        Line  (FLPy)                   (moFLP)                        FRT) 3703                                 3953                                     pUC18                                         Note    __________________________________________________________________________    1   GLEAN ®              0.0001 μg           10.0 μg    2   Resistant              0.001 μg            10.0 μg    3   BMS-  0.01 μg             10.0 μg    4   FRT   0.1 μg              9.9 μg    5   Transfor-              1.0 μg              9.0 μg    6   mants 10.0 μg    7              0.0001            10.0 μg                   μg    8              0.001 μg       10.0 μg    9              0.01 μg        10.0 μg    10             0.1 μg         9.9 μg    11             1.0 μg         9.0 μg    12             10.0 μg    13                  0.0001 μg 10.0 μg    14                  0.001 μg  10.0 μg    15                  0.01 μg   10.0 μg    16                  0.1 μg    9.9 μg    17                  1.0 μg    9.0 μg    18                  10.0 μg    19                       10.0 μg    20  BMS                      0.1 μg                                     9.9 μg    21  Control              1.0 μg    22             1.0 μg    23                  1.0 μg    24  Glean ®                  Un-shot        Resistant                        Control        BMS-        FRT        Transfor-        mants    25  BMS                              Un-shot        Control                          Control    __________________________________________________________________________

GUS activity indicating FLP-mediated excision of genomic DNA in cellstransfected with FLYy or moFLP is shown below in Tables II-D-2 andII-D-3. The data demonstrate that the ED₅₀ and maximal response whenmoFLP is used is approximately 5-fold greater than when FLPy is used toexpress FLP. Significantly, the same number of excision seen withoptimal levels of the Ubi::FLPy plasmid were produced with 100-foldlower plasmid dosage using Ubi::moFLP.

                  TABLE II-D-2    ______________________________________    Treatment #   Mean (GUS)   Standard    (n = 3)       Blue foci per plate                               Deviation    ______________________________________    1 FLPy        0            0    2             0.67         0.58    3             10.33        9.71    4             144.3        19.43    5             321.67       43.41    6             273.67       62.13    7 moFLP       11.33        3.51    8             66           22.54    9             262.67       31.63    10            984          148.25    11            1245.67      177.75    12            686.33       93.51    13 moFLP/FRT  0.67         1.15    14            8            3.61    15            57.33        18.34    16            493          85.07    17            1073.33      100.42    18 Controls   214          174.79    19            0            0    20            1086         149.66    21            0            0    22            0            0    23            0.33         0.58    24            0            0    25            0            0    ______________________________________

The ED₅₀ for each FLP expression plasmid shown in Table II-D-3 wasmanually determined from graphing the data.

                  TABLE II-D-3    ______________________________________    FLP expression vector                  ED.sub.50 (μg FLP expression vector)    ______________________________________    PHP5095 FLPy  0.13 μg    PHP5096 moFLP 0.03 μg    PHP5849 MoFLP/FRT                  0.11 μg    ______________________________________

E. FLP Excision of Genomic DNA Sequences

To further evaluate site-specific excision of DNA sequences by moFLP,BMS cell lines were transformed with plasmids carrying moFLP and FRTsequences, using the methods described above for Examples I and II.Specifically, PHP5096 carrying moFLP (described above for Example I-A,see Table I-A-1) and PHP5954 carrying moFLP and FRT sequences. TheFRT-containing control PHP3456 is described above for Example I-F, seeTable I-F-1). The experimental design is shown below in Table II-E-1.Carrier DNA control PHP3703 is described above for Example II-D. Tonormalize values for GUS expression (turned on by FLP-mediated excisionof an intervening sequence between promoter and structural gene) aconstitutively expressed luciferase plasmid (PHP4992; UBI:Ubiintron:luciferase:pinII) was introduced at a constant amount in alltreatments.

                                      TABLE II-E-1    __________________________________________________________________________        PHP  PHP PHP  PHP  PHP    Treat-        4992 3456                 3703 5096 5954    ment #        (control)             (FRT)                 (control)                      (moFLP)                           (moFLP/FRT)                                   Notes    __________________________________________________________________________    1                              Un-shot cells (negative control)    2   1. μg             7. μg                 2. μg          Negative control of GUS                                   activity    3   1. μg             7. μg                 2. μg                      0.001 μg    4   1. μg             7. μg                 2. μg                      0.01 μg    5   1. μg             7. μg                 1.9 μg                      0.1 μg    6   1. μg             7. μg                 1. μg                      1. μg    7   1. μg             7. μg 2. μg                           0.001 μg    8   1. μg             7. μg 2. μg                           0.01 μg    9   1. μg             7. μg 1.0 μg                           0.1 μg    10  1. μg             7. μg 1. μg                           1. μg    11  1. μg             7. μg      1. μg                                   Negative control (for GUS                                   activity in PHP5954).    12       2.5 μg     1. μg                                   To compare with treatment    __________________________________________________________________________                                   #14

The transformation, cell culture and assay methods were as describedabove for Example I. A GUS chemiluminescent assay was used following themanufacturer's protocol. In general, crude extract was incubated for onehour with Glucuron substrate at room temperature. The incubated cuvettewas then inserted into an Analytical Luminescence Laboratories Model#2010 Monolight luminometer which was set to inject 100 TI Acceleratorsolutions, wait five seconds, then count the emitted light. The data areshown below in Table II-E-2, and are expressed as net GUS Relative LightUnits (RLU) per net luciferase RLU times 10³.

                  TABLE II-E-2    ______________________________________           Response  (Net GUS           R.L.U./Net Luciferase R.L.U.)*10.sup.3 !    Treatment #      Standard    (n = 3)  Mean    deviation Note    ______________________________________    1        278     240       Negative control baseline.    2        -15.7   22.6      Negative control    3        283     373    4        4338    759    5        11496   1092    6        9049    2443    7        28.3    64.4    8        1007    217    9        3226    1438    10       13521   2241    11       -6.3    2.9    12       2327    261    ______________________________________

moFLP expressed recombinase effectively excised DNA sequences toactivate GUS (treatnents 3-6). As expected, no GUS activity wascontributed by PHP5954 (treatment #11). FLP activity from transientexpression of PHP5954 was shown by excision from co-delivered PHP3436(treatments 7-10).

Example III Transgenic Maize Cells Expressing FLP Activity

Healthy, actively growing maize cells with moFLP integrated in thegenome are shown to express moFLP resulting in proper FLP-mediated sitespecific excision.

A. Production of FLP-containing Transformants.

A transginic maize cell line expressing FLP was prepared by insertingmoFLP nucleic acid sequences into BMS cells, using materials, methods,and plasmids described above or Examples I and II. The moFLP-containingplasmid PHP5954 described above for Example II-E (see Table II-E-1) andthe BAR-expressing control plasmid PHP3528 described above for ExampleII-C (see Table II-C-1) were used in these studies. Cells weretransferred to fresh medium two days prior to bombardment, andresuspened in osmoticum one day prior to bombardment. A mass of 5 ng ofeach plasmid to co-transform cells.

Post-bombardment, transformants were selected for BAR expression onmedium #306 containing 5 mg/ml Basta (medium #306E containing Bastacommercial herbicide formulation from Agrevo, containing the activeingredient ammonium glufosinate). More than 100 Basta-R transformantswere recovered. These were analyzed for FLP activity as described belowin III-B.

B. Identification of FLP Expressing Events among Basta-R Transformants

Independent BMS Basta-R transformants produced in Example III-A weremaintained on #306 E medium. To screen for FLP expression and activity,transformed cells were transfected with PHP3456, which contains a 1.8 kbintervening sequence between direct FRT sites (see Table I-F-1). UponFLP-mediated excision of the intervening sequence, GUS is expressed fromthe plasmid in host cells.

At three days before bombardment, transformed cells (approximately25-100 mg of each Basta-R transformant) were placed on paper filters (4events/filter) on top of plates of #306 medium and incubated understandard conditions, as described above for Example II-B (27° C., nolight). On the day of bombardment, each filter with cells was brieflydried by vacuum filtration to remove excess moisture from the filter,and then placed onto a second, dry filter in an empty Petri dish.Osmoticum (#586 medium+0.25 M sorbitol) was added and the samples wereincubated for five hours before bombarding.

Bombardment conditions and methods were as described above for ExamplesI and II. The plasmids used included the FLP and FRT-containing PHP5954,described above for Example II-E, and PHP5096 expressing moFLP,described above for Example I-A (see Table I-A-1) as positive controls.PHP3456 expressing GUS on FLP-mediated excision is described above forExample I-F (see Table I-F-1). PHP3457, a positive control for GUSactivity, is described above in Table I-F-1.

After bombardment, plates were incubated overnight at 27° C., withoutlight, and then stained with 1 mL X-gluc buffer per plate, as describedabove for Examples I and II.

The experimental design is shown below in Table III-B-1.

                                      TABLE III-B-1    __________________________________________________________________________        BMS Cell lines        (n = 5 plates; with 20        independent Basta-    Treat-        Resistant   PHP                       PHP PHP    ment #        Transformants/plate)                    3456                       5096                           3457                              Notes    __________________________________________________________________________    1   moFLP/FRT,  5 μg   Express GUS on FLP-        Basta-R Transformants mediated excision    2   moFLPIFRT,  5 μg        Basta-R Transformants    3   moFLP/FRT,  5 μg        Basta-R Transformants    4   moFLP/FRT,  5 μg        Basta-R Transformants    5   moFLP/FRT,  5 μg        Basta-R Transformants    6   Non-transgenic control                    5 μg   Negative control    7   Non-transgenic control                    5 μg                       1.5 μg                              Positive control FLP    8   Non-transgenic control                           1 μg                              Positive control GUS    __________________________________________________________________________

Out of 100 Basta-resistant transformants obtained in Example III-A, 14were GUS positive, indicating FLP activity:

    ______________________________________    #Blue foci/event                    Number of    (GUS+)          events    ______________________________________     1-10           3    11-50           3     51-100         6    >100            2    ______________________________________

This result provided the first evidence that transformed maize cellsexpressing moFLP could actively grow (divide). Positive cell linesprovided hosts for retransformation, by delivery of a promoterless FRTcoding sequence fusion for site-specific recombination at the FRT in thetransgenic PHP5954 sequence in these cell lines.

C. Maize Cell Line Expressing FLP and Carrying an FRT Site forSelf-Inactivation

To obtain BMS transformants carrying moFLP and FRT sites, BMS cells werebombarded with DNA particles carrying the FRT/FLP-containing plasmidPHP5954 described above for Example II-E. The structural gene, moFLP wasdesigned to express until a FRT:: non-FLP! sequence is recombined at theFRT site of the transgene. The control plasmid PHP3528 expressing BAR,described above for Example II-C (see Table II-C-1), was used todetermine DNA transformation efficiency.

After bombardment, transformed cells were screened for resistance tobialophos (Basta-R). A total of 91 Basta-R events were isolated. Basta-Rtransformants were maintained on medium #306E, and then screened for FLPexpression, as described below in III-D.

D. Screening BMS-FLP/FRT Transformants for FLP Activity

To screen the BMS-moFLP/FRT Basta-R transformants produced in ExampleIII-C for FLP activity, transformed cells were transfected with PHP3456using the procedure described above for Example III-B. On FLP-mediatedexcision of the 1.8 kb intervening sequence, GUS is expressed. Controlplasmid PHP5954 is described in Example II-C. The silent reporterPHP3456 expresses GUS on FLP-mediated excision and is described above inExample I-F (see Table I-F-1). The control plasmid PHP687 expressing Rand C 1 was used to access bombardment efficiency and is described abovein Example II-B. The experimental design is shown below in TableIII-D-1. For treatments 1 to 4, 5 plates were used per treatment with 4independent transformants per plate. For treatment 5, 2 plates with 3independent transformants per plate. Treatment 6-8 used non-transgeniccontrol callus.

                                      TABLE III-D-1    __________________________________________________________________________    Treat-        PHP PHP PHP    ment #        BMS Cell Lines                  3456                      5954                          687 Notes    __________________________________________________________________________    1   BMS-moFLP/FRT                  5 μg                      --  0.01 μg        Basta-R    2   BMS-moFLP/FRT                  5 μg                      --  0.01 μg        Basta-R    3   BMS-moFLP/FRT                  5 μg                      --  0.01 μg        Basta-R    4   BMS-moFLP/FRT                  5 μg                      --  0.01 μg        Basta-R    5   BMS-moFLP/FRT                  5 μg                      --  0.01 μg        Basta-R    6   Control (n = 12)                  --  --  --  Non-transgenic (non-FLP) negative                              control    7   Control (n = 12)                  5 μg                      1 μg                          0.01 μg                              Positive control    8   Control (n = 12)                      1 μg                          0.01 μg                              Negative GUS control for                              treatment #7    __________________________________________________________________________

Out of 86 independent Basta-R transformants, eight gave blue spots(GUS+) indicating FLP expression and activity. This experimentdemonstrates a viable BMS cell line expressing FLP and having FRT sites.

E. Co-Expression of FLP and a Reporter

The expression of a reporter protein from a plant transcription unitcontaining an FRT site at its N-terminus (PHP3457) was examined in thepresence of a co-transfected plasmid expressing moFLP (PHP5096) in theembryogenic suspension cell line, 3-44-6E1. Cell culture and bombardmentprocedures were performed as described for Example I-E. Luciferase andGUS chemiluminescent assays were performed as described above forExamples I and II.

Plasmids used for these studies included the FLP-expressing PHP5096,described above for Example I-A (see Table I-A-1), PHP3457, theGUS+reporter plasmid having an FRT site at its N-terminus, describedabove for Example I-F (see Table I-F-1), PHP3703, a non-GUS, non-FLPnegative control plasmid as described above for Example II-D (see TableII-D-1). A second luciferase reporter was used in this experiment tonormalize DNA delivery across treatments, PHP1528, which contained theluciferase structural gene driven by the Enhanced CaMV35S promoter withOmega' and the Adh1 intron I 5' to the gene and the pinII 3' region. Theexperimental design os shown below in Table III-E-1.

                                      TABLE III-E-1    __________________________________________________________________________    Treatment #          PHP1528               PHP3703                    PHP5096                         PHP3457                              Notes    __________________________________________________________________________    1     1. μg               1.1 μg      Negative GUS control.    2     1. μg               0.1 μg                    1. μg  Negative GUS control.    3     1. μg               1. μg  0.1 μg                              Positive control; benchmark for                              inhibition assay    4     1. μg  1. μg                         0.1 μg    __________________________________________________________________________

One day after bombardment, extracts were prepared and assayed for GUSand luciferase activities. After subtracting background activities forGUS and luciferase from each sample, GUS activities were then divided byrespective luciferase activities, in order to normalize for variation inthe efficiency of DNA delivery among replicates nd treatmints. Thenormalized data is shown below in Table III-E-2.

                  TABLE III-E-2    ______________________________________    Treatment #     Mean    Standard    (n = 3)         GUS+    deviation    ______________________________________    1               -11.5   1.7    2               -30.0   11.0    3               412.1   23.6    4               470.7   18.7    ______________________________________

These data demonstrate transient expression and activity of FLP does noteffect co-expression of a reporter containing an FRT site fused to itscoding sequence.

Example IV FRT Recombination in Regenerable Maize Callus and Plants andExpression of Modified FLP Encoded Recombinase in Maize Callus andPlants

A. FLP and FRT Plasmid DNA Construction

FLP vectors were constructed using standard molecular biology techniques(Sambrook et al. eds.). The plasmid PHP8007 is described, but the othervectors used in these experiments contain combinations of the same basicregulatory elements. Plasmid PHP8007 contains a selective marker gene,bar (Thompson et al. (1987) EMBO J 6:2519-2523) driven by a CauliflowerMosaic Virus 35S promoter with a duplicated enhancer region (Gardner etal. (1981) Nucl. Acid Res. 9:2871-2888). The 79 bp Tobacco Mosaic Virusleader (Gallie et al. (1987) Nucl. Acid Res. 15:3257-3273) was inserteddownstream of the promoter followed by the first intron of the maizealcohol dehydrogenase gene ADH1-S (Dennis et al. (1984) Nucl. Acid Res.12:3983-3990). The pinII terminator was ligated downstream to create theBAR expression cassette. PHP8007 also contains the modified FLP genedriven by the promoter region of the maize ubiquitin gene Ubi-1(Christensen et al. (1992) Plant Mol. Biol. 18:675-689). The pinIIterminator was ligated downstream to create the FLP expression cassette.PHP8007 and other FLP and FRT-containing plasmids used in transformationof embryogenic maize events are listed in Table IV-A-1 below.

                                      TABLE IV-A-1    __________________________________________________________________________    Plasmids used to verify FLP activity and FRT sites in regenerable maize    callus,    regenerated plants and progeny.                     Structural                          3'    PHP Promotor             5' intron                     Gene sequence                               Purpose    __________________________________________________________________________    Plasmids containing FLP recombinase expression-cassette;    5096        UBI  Ubi-intron                     moFLP                          pinII                               Maize-optimized    5954        UBI  Ubi-intron/FRT                     moFLP                          pinII                               Maize-optimized    8007        UBI  Ubi-intron                     moFLP                          pinII                               Maize-optimized    Plasmids Containing FRT sequences to demonstrate excision of bar and    activation of GUS.    5869        Enhanced             adhI-intron/FRT                     bar  pinII/FRT                               Excision of bar gene from        35S               :GUS::pin                               stably integrated genomic                          II   locus    __________________________________________________________________________

B. Preparation of Callus with Integrated FRT-containing Sequences

Hi-II germplasm, a maize genotype with a high frequency and vigor oftype II culture initiation was selected out of an A188×B73 cross (seeArmstrong et al. (1991) Maize Genetics Newsletter 65:92-93) and ispublicly available.

FRT recombination in regenerable maize callus and plants was evaluatedby the stable transformation of the plasmid PHP5869 into embryogeniccallus cultures of the genotype Hi Type II (Hi-II). Rapidly growingembryogenic callus was suspended in liquid medium and sieved through a860 urn screen. Approximately 250 mg fresh weight of sieved callus wasthen suspended in 5 ml of medium and evenly distributed onto a 5.5 cmglass fiber filter disk using vacuum filtration on a 4.7 cmmicroanalysis holder. DNA was delivered into the cells using the BioradPDS-1000 biolistics apparatus. Precipitation of plasmid DNA onto 1.0 umtungsten particles was as described by Klein et al. (1988) Proc. Nat.Acad. Sci. 85:4305-4309. Post-bombardment selection for cells expressingthe bar gene was done essentially as described in Register et al. (1994)Plant Mol. Biol. 25:951-961; following bombardment, the filter disk andcells were transferred to callus culture medium for 7 days after whichthe filter disk and cells were transferred to fresh callus culturemedium containing 3 mg/l bialaphos (callus culture medium andregeneration media were similar to those described by Armstrong (1994)in "The Maize Handbook", Freeling and Walbot, eds. Springer-Verlag, NewYork, pp663-671). After another 7 days the cells were removed from thefilter disk and suspended in 5 ml of selection medium (held at 37° C.)containing 0.6% (w/v) low melting point agarose (Sea-Plaque; FMC, Inc.).The suspension was divided into 2 equal aliquots and each aliquot wasevenly plated over solidified medium in 100×15 mm Petri dishes. After4-6 weeks, rapidly growing putative transformed calli were removed andtransferred to the surface of fresh selection medium.

The accessibility and finctionality of the FRT sites was evaluated bytransiently expressing FLP recombinase in the callus using particledelivery of the recombinase DNA. Cell preparation and DNA delivery wasas described above. The target sequence contained in PHP5869 wasconstructed such that recombination of the FRT sites within the sequencewould result in excision of the bar gene and expression of the scorablemarker GUS. A total of 25 putative events containing PHP5869 were testedfor FRT recombination. The recombination potential of the events wasscored by visual observation of the number of GUS-expressing foci whichcould be observed after exposure of the cells to GUS substrate 3 daysafter bombardment. The results are summarized in Table IV-B-1 below.

For relative level of recombination with PHP5869 events:

                  TABLE IV-BA-1    ______________________________________    Recombination                 None(-) +        ++   +++    ++++    Score    Percent of Events                 32%     8%       12%  36%    12%    ______________________________________     + = 1 to 5 blue spots; ++ = 5-25; +++ = 25-100; ++++ = >100 spots per     plate

As the data above demonstrate, a high percentage of transformationevents exhibited high levels of FLP-induced excision. Of the events thatexhibited recombination, 12 were regenerated for analysis at the wholeplant level (Table IV-B-2). For events exhibiting FLP-mediatedrecombination that were regenerated for further analysis:

                  TABLE IV-B-2    ______________________________________    Event Number  Recombination Score    ______________________________________    776.71-47-1   +++    776.77-68-1   ++    776.83-19-1   ++    776.85-21-1   +++    776.89-17-4   ++++    776.89-17-6   +++    776.89-17-7   +++    776.89-19-1   ++++    776.89-19-3   +++    776.89-19-5   ++++    776.89-19-10  +++    776.89-20-1   +++    ______________________________________

C. Evaluation of FRT Events in Progeny

A second set of tests were performed to evaluate PHP5869 stabletransformed calli (from progeny seed, but using the method describedabove) and progeny embryos. These calli and embryos derived from thedifferent transformation events were bombarded with PHP8007 plasmid DNA.Transient FLP gene expression (from plasmid PHP8007) catalyzed excisionof bar and resulted in activation of the GUS gene in PHP5869 stabletransformed cells. The level of GUS expression and the density of GUSstaining in these cells were used to evaluate the "quality" of PHP5869transformed events. The results are reported in Table IV-C-1. Forresults of bombardment of PHP6869 transformants with PHP8007:

                                      TABLE IV-C-1    __________________________________________________________________________               Embryo Bombardment                               Callus Bombardment                          GUS             GUS    Event No.          Pedigree               Total                   GUS+                       %  Density                               Total                                   GUS+                                       %  Density    __________________________________________________________________________    776.89-17-6          T0xGS3               40  3    8%                          +    6   4    67%                                          +    776.89-17-7          T0xGS3               143 32  22%                          +    25  13   52%                                          ++    776.89-17-7          T0 self               38  10  26%                          ++   18  3    17%                                          ++    776.89-19-1          T0xGS3               54  28  52%                          +++  11  10   91%                                          +++    776.89-19-3          T0xGS3               39  9   23%                          +++  23  21   91%                                          ++    776.89-19-3          T0 self               30  9   30%                          +++  3   3   100%                                          ++    776.89-19-5          T0xGS3               47  31  66%                          +++  67  60   90%                                          +++    776.89-19-5          T0 self               92  36  39%                          +++  25  21   84%                                          +++    776.89-20-1          T0xGS3               57  19  33%                          +++  16  15   94%                                          ++    776.89-20-1          T0 self              15  15  100%                                          ++    __________________________________________________________________________

The bombardment results shown in Table IV-C-1 confirmed three things: 1)FLP recombinase can catalyze site-specific recombination at FRT sites inthe maize genome, the functionality of FRT sequences is heritable inmaize, and 3) there are differences in recombination frequency and GUSexpression between FRT events. These difference may reflect the"quality" of the FRT events for site specific recombination, and werealso observed in the F1's of FRT×FLP (see below).

D. FLP Gene Transformation and Evaluation of FLP Activity

Transformation of the FLP plasmid DNA, PHP8007, in Hi-II followed thestandard Hi-II bombardment transformation protocol (Songstad D. D. etal. (1996) In Vitro Cell Dev. Biol. Plant 32:179-183). Cells weretransformed by culturing maize immature embryos (approximately 1.5 mm inlength) onto 560P medium containing N6 salts, Erikkson's vitamins, 0,69g/l proline, 2 mg/l 2,4-D and 3% sucrose. After 4-5 days of incubationin the dark at 28° C., embryos were removed from 560P medium andcultured, coleorhizal end up, onto 560 L medium which is equivalent to560P but contains 12% sucrose. Embryos were allowed to acclimate to thismedium for 3 h prior to transformation. Embryos were transformed usingthe PDS-1000 Helium Gun from Bio-Rad at one shot per sample using 650PSIrupture disks. DNA delivered per shot averaged at 0.0667 ug. Followingbombardment, all embryos were maintained on 560L medium for 48 hoursbefore transfer to 3% sucrose and 3 mg/l bialaphos. Plates weremaintained at 28° C. in the dark and were observed for colony recoverywith transfers to fresh medium occurring every two weeks. Transgeniccolony recovery was noted initially as growing callus tissue with ahealthy phenotype on selection.

The transformed calli were evaluated for FLP activity, callus morphologyand plant regeneration ability to determine whether the FLP activityaffects callus formation and plant regeneration.

FLP activity was evaluated by bombarding PHP5869 plasmid DNA intoPHP8007-transformed calli. The bombarded calli were cultured for 2 to 3days under normal culture condition and then stained with X-gluc toscore GUS gene expression. If the FLP gene was expressing, it wouldrecombine the FRT sites in plasmid PHP5869, resulting in excision of barand connection of the GUS gene to the 35S promoter to activate GUS. Ifthe FLP gene was more active in a particular transformed callus, moredark blue cells would be observed.

Callus morphology was evaluated by examining the proportion of somaticembryos in each of the embryo-derived calli. Callus containing moresomatic embryos would be capable of generating more plants.

The ability of calli to regenerate plants was evaluated by tabulatingthe number of plants produced from each of the embryo-derived calli. Asingle embryo-derived callus producing more than 15 plants was given thehighest score. Progressively lower scores were given to calli producing11-15 plants, 6-10 plants, 1-5 plants or no plants. Each characteristicwas evaluated using a scale with 4 representing the best (or highest)rating and 1 representing a low level, while 0 represented an absence ofthat characteristic. Results of this evaluation are reported below inTable IV-D-1.

For evaluation results of FLP transformed calli:

                  TABLE IV-D-1    ______________________________________                        Plant regeneration    FLP   Total   Callus morphology    Event    Activity          Events  Score  Event No.                                %     Score                                           No.   %    ______________________________________    4     30      4      4      13.3% 4    7     23.3%                  3      5      16.7% 3    3     10.0%                  2      10     33.3% 2    3     10.0%                  1      11     36.7% 1    7     23.3%                  0      0      0.0%  0    10    33.3%    3     21      4      1      4.8%  4    2     9.5%                  3      2      9.5%  3    4     19.0%                  2      13     61.9% 2    3     14.3%                  1      5      23.8% 1    8     38.1%                  0      0      0.0%  0    4     19.0%    2             4      0      0.0%  4    0     0.0%                  3      2      22.2% 3    0     0.0%                  2      1      11.1% 2    0     0.0%                  1      6      66.7% 1    1     11.1%                  0      0      0.0%  0    8     88.9%    1     11      4      0      0.0%  4    0     0.0%                  3      3      27.3% 3    2     18.2%                  2      4      36.4% 2    1     9.1%                  1      4      36.4% 1    1     9.1%                  0      0      0.0%  0    7     63.6%    0     11      4      2      18.2% 4    4     36.4%                  3      3      27.3% 3    1     9.1%                  2      5      45.5% 2    2     18.2%                  1      1      9.1%  1    3     27.3%                  --     --     --    0    1     9.1%    ______________________________________

Even in events not expressing FLP (category 0 under FLP activity), awide range of callus morphologies and regeneration capacities wereobserved. As FLP activity increases to the highest level observed, thevariability in callus quality and regeneration appears to remain highand no apparent correlation can be discerned.

To verifiy the effect of the FLP activity on transgenic plant fertility,pollen morphology and female gamete fertility were evaluated. Pollenmorphology was used as an indicator for pollen viability. The pollenmorphology results are reported in Table IV-D-2. The results in TableIV-D-2 demonstrate that FLP activity had some negative impact on pollenviability. For events with higher FLP activity in T0 plants, thepercentage of events with normal pollen was lower. However, the dataalso showed that in the events with highest (4) FLP activity in T0plants, there were still 26.7% of the transgenic events whose pollenappeared normal.

                                      TABLE IV-D-2    __________________________________________________________________________            Plants with Normal                      Plants with 50%                               Plants with Mixed    FLP No. of            Pollen    Normal Pollen                               Pollen*    acitivity        Events            Event No.                 %    Event No.                           %   Event No.                                    %    __________________________________________________________________________    4   12  4    26.7%                      9    60.0%                               2    13.3%    3   12  6    50.0%                      5    41.7%                               1    8.3%    2   0   0         0        0    1   1   1         0    0.0%                               0    0.0%    0   6   3    50.0%                      0    0.0%                               3    50.0%    __________________________________________________________________________     *Plants with mixed pollen is a group of T0 plants that were regenerated     from the same embryoderived callus. Some plants showed normal pollen and     others had 50% normal pollen. Southern analysis showed that the plants     with different morphologies of pollen represent different transformation     events.

Female gamete fertility can be determined by observing T1 seed formationfollowing crosses made between FLP T0 plants as the female parent andnon-transformed plants as the male parent. If the FLP-containing femalegamete showed normal inheritance, about 50% of the T1 seeds shouldcontain the FLP gene. If FLP expression has a negative impact on femalefertility, less than 50% of the T1 seeds should have the FLP gene. FLPactivity in the T1 seeds was assayed by bombarding PHP5869 into T1embryos as described above for FLP callus bombardment. T1 seedbombardment results are reported in Table IV-D-3.

For FLP T1 seeds bombardment with PHP5869:

                  TABLE IV-D-3    ______________________________________    FLP                     Total    activity       Pollen   seeds  Seeds with                                           Seeds with    in T0  Total   Morpho-  bom-   FLP+    FLP-    generation           events  logy     barded No.  %    No.  %    ______________________________________    4      3       normal   300    146  49%  154  51%    4      6       50%      226    48   21%  178  79%                   normal    ______________________________________

As shown in Table 6, the 300 seeds derived from 3 events that havenormal pollen showed 49% FLP+ and 51% FLP-. This segregation is veryclose to Mendelian 1:1 ratio. In these events, the FLP activity does notreduce the fertility of the female gametes. On the other hand, the 226seeds derived from 6 events that have 50% normal pollen showed 21% FLP+and 79% FLP-. In these events, the FLP activity does reduce thefertility of the female gametes. In addition, an average of 130 seedswere formed on each of the 5 T0 plants (3 events) that have normalpollen, while an average of 20 seeds were formed on each of the 14 T0plants (6 events) that have 50% normal pollen. In non-transformed Hi-II,there are usually 100-150 seeds formed on most of the ears. These dataconfirmed that the kernel formation (female gamete fertility) is notreduced in those T0 plants with normal pollen, but is reduced in thoseT0 plants with 50% normal pollen.

Combining the data in both Table IV-D-2 and Table IV-D-3, it appearedthat FLP activity influenced both male and female gametes in maize, andthis effect was usually observed in the same plants. However, thiseffect does not reduce the utility of the FLP system in maize becauseabout 27% of the transgenic events showed high FLP activity and bothmale and female gametes were normal.

These data suggested that evaluation of plant fertility inFLP-containing events before using them is one of the key steps forsuccessful application of this system in maize. If negative effectsoccur in certain plants, it will usually affect both male and femalegametes. We can use the T0 pollen evaluation technique to quickly andeasily determine the affected and non-affected plants.

DNA from T0 plants recovered following stable transformation with theFLP recombinase gene was subjected to Southern analysis using standardtechniques. Many of the plants contained DNA bands corresponding to bothFLP and bar genes. FLP activity appeared to be related to Southernpatterns. Usually, events with a low copy number of the gene and asimple insertion pattern gave rise to high FLP activity. However, thecorrelation between Southern patterns and the effect of FLP activity onthe fertility of gametes is not clear.

E. Evaluation of the F1 Generation

The purpose of making F1 crosses between FLP plants and FRT plants is tobring FLP and FRT together in the same genome and then to evaluate FRTsite-specific recombination catalyzed by FLP recombinase. FRTsite-specific recombination results in the excision ofFRT::bar::PIN-II::FRT and activation of a GUS gene. Thus, the GUS genewas used as the visible marker to identify FLP recombinase catalyzedsite-specific recombination in the F1. F1 crosses were made using eitherFLP-expressing plants as a female and FRT/bar/GUS-containing plants asthe male. Reciprocal crosses were made also. F1 seeds were collectedfrom these crosses and among 1,412 germinated plants, 1031 plants or 73%of the total plants were Ignite positive, indicating expression of thebar gene (tested by painting a 2% solution of Ignite onto the leaves;Ignite is a commercial herbicide from AgrEvo containing the activeingredient ammonium glufosinate). This value is very close to the 75%expected from 3:1 segregation. Among those 1,412 plants, theoretically,25% or 353 plants should contain both FLP recombinase and FRT/Bar/GUSDNA in the same plants. Only these 353 plants containing both DNAsequences (a FLP expression cassette and FRT sites) could potentiallycatalyze site-specific recombination to activate GUS. Leaf tissuescollected from all Ignite positive plants were stained with X-gluc toassay for GUS expression. 197 plants expressed GUS. The range of levelsof GUS expression in F1 plants ranged from no expression to high levels,and varied from event to event. On average, the frequency was 55.8%.These results demonstrated that the nucleic acid sequence encoding FLPcan be transmitted and is active in subsequent generations forsite-specific recombination. Furthermore, events containing a genomicFLP expression cassette and genomic FRT sites can be crossed to effectexcision of the FRT-flanked sequence.

All GUS expressing F1 plants were subjected to PCR analysis to confirmbar gene excision. One primer was designed within the 35S promoterregion and a second primer was within the GUS gene. Excision of the bargene should yield a PCR fragment of 250 bp, as compared to a 1350 bpexpected if the bar gene is not excised. The PCR assays confirmed thatall GUS expressing F1 plants had a 250 bp band.

In the F1 crosses, only the events with high FLP activity (scored as 3and 4, see Table IV-D-1) that had normal pollen in their T0 plants wereused and the recombination frequency mainly depended on the quality ofFRT events. Some comparisons of the recombination frequency in differentcrosses are listed below in Table IV-E-1. When FLP event 260891 was usedfor the F1 crosses, different FRT events used in the F1 crosses resultedin different recombination frequencies. For example, when FRT events776.89-19-3 and 776.89-20-1 were used, the recombination frequency was100% in the F1 plants, while if 776.89-17-6 and 776.89-17-7 were usedfor the F1 crosses, the recombination frequencies were about 40% in theF1 plants. If FRT event 776.89-19-1 was used for the F1 cross, norecombination was observed in the resulting F1 plants.

For recombination frequency in different F1 crosses:

                  TABLE IV-E-1    ______________________________________                        Recombination frequency in F1    FLP Event FRT event cross    ______________________________________    260891    776.89-17-6                        41%    260891    776.89-17-7                        40%    260891    776.89-19-1                        0%    260891    776.89-19-3                        100%    260891    776.89-19-5                        92%    260891    776.89-20-1                        100%    ______________________________________

In the F1 crosses, both FRT and FLP were used as either male or femaleperents. The evaluation results demonstrated that in general, therecombination frequency in the F1 plants was higher if FLP events wereused as the female parent, as opposed to using the FRT events as thefemale parent. Table IV-E-2 provides a few examples of this comparison.

For recombination frequency in the reciprocal crosses between FLP andFRT:

                  TABLE IV-E-2    ______________________________________    Event as Female              Event as Male in F1                           F1 Plants    Crosses   Crosses      Ignite.sup.+                                   Gus.sup.+                                        Recombination    ______________________________________    FRT/776.89-19-1              FLP/260935   22      1    14%    FLP/260935              FRT/776.89-19-1                           28      7    75%    FRT/776.89-19-3              FLP/260935   12      3    75%    FLP/260935              FRT/776.89-19-3                           13      0    0    FRT/776.89-19-5              FLP/260935   31      1    10%    FLP/260935              FRT/776.89-19-5                           39      6    46%    FRT/776.89-20-1              FLP/260935   25      4    48%    FLP/260935              FRT/776.89-20-1                           34      8    71%    FLP as male            90      9    30%    FLP as female          114     21   55%    ______________________________________

In Table IV-E-2, Ignite+ indicates Ignite resistant plants among the F1plants. Theoretically, one third of the Ignite resistant F1 plantscontained both FLP and FRT constructs in the same plants. Only thisfraction (1/3) that contain both FLP and FRT had the potential forsite-specific recombination. The recombination frequency in the lastcolumn in Table IV-E-2 was calculated based on this estimate. The datain Table IV-E-2 demonstrated that the recombination frequency was higherwhen the FLP containing events were used as the female rather than asthe male (in 3 out of 4 F1 crosses). On average, the recombinationfrequency was 55% in the crosses where FLP expressing plants were usedas the female parents, while the frequency was 30% in the reciprocalcrosses. One explanation is that in maize, as in most plants, thecytoplasm in the zygote is inherited from the female gamete, and spermcells carry little cytoplasm to the fertilized zygotes. In the casewhere FLP expressing plants were used as a female parent, the existingFLP recombinase made in the female cytoplasm may bind to the FRT sitesbrought in by male gametes immediately and catalyze excision rapidly. Inthe reciprocal case FLP protein synthesis would have to start anew afterfertilization, thereby delaying excision. In the GUS expressing F1plants, uniform GUS staining of the leaf tissues collected from threeleaves in a single plant was common, although some chimeric GUSexpressing plants were observed. Uniform GUS staining indicated that thesite-specific recombination occurred most likely at the single cellstage of the zygote while the chimeric GUS expressed plants could resultfrom either later bar gene excision or GUS gene silencing (Flavell(1994) Proc. Natl. Acad. Sci. USA vol. 91, 3490-3496); Matzke and Matzke(1995) Plant Physiol. 107:679-685); Matzke and Matzke (1995) TIG, vol.11, no. 1, 1-3). These two possibilities can be distinguished by PCRassays. If both GUS-expressing tissue and non-GUS expressing tissue fromthe same plants contain the 250 bp PCR band, this non-GUS expressingsector(s) is likely the result of GUS gene silencing. On the other hand,if the 250 bp PCR band can be found in the GUS expressing sector(s), anda 1,350 bp band is found in the non-GUS expressing sector(s), thischimera is due to later recombination in this plant.

F. Evaluation of the F2 Generation

The F1 plants expressing GUS were pollinated by non-transformed pollento generate F2 seeds. The F2 generation was evaluated for segregation ofthe genes. Normally, four different genotypes are expected in the F2,i.e. FLP+/BAR+, GUS+/BAR-, FLP+/BAR+/GUS+ and FLP-/BAR-/GUS- and theyshould segregate in a 1:1:1:1 ratio. These four genotypes werephenotypically either Ignite+ or Bialaphos+/GUS- (FLP+/BAR+), Ignite- orBialaphos-/GUS+ (GUS+/BAR-), Ignite+ or Bialaphos+/GUS+ (FLP+/BAR+/GUS+)and Ignite- or Bialaphos-/GUS- (FLP-/BAR-/GUS-). The F2 embryos wereplaced on medium for callus initiation and then the callus from eachembryo was divided into three parts, one was used for GUS staining toverify the GUS expression, one was used for culturing on Bialaphoscontaining medium to verify bar expression and one was kept on the samemedium used for callus initiation. Embryos from 4 F1 plants were assayedwith this method and the results are reported in Table IV-F-1.

For F2 embryo segregation:

                                      TABLE IV-F-1    __________________________________________________________________________              GUS+/  GUS-/  GUS+/  GUS-/    F1  Total F2              Bialaphos+                     Bialaphos+                            Bialaphos-                                   Bialaphos-    Plant        embryos              No.                 %   No.                        %   No.                               %   No.                                      %    __________________________________________________________________________    27-14        69    17 24.6%                     15 21.7%                            19 27.5%                                   18 26.1%    28-10        54    8  14.8%                     13 24.1%                            11 20.4%                                   22 40.7%    28-13        46    9  19.6%                     7  15.2%                            14 30.4%                                   16 34.8%    33-10        92    28 30.4%                     21 22.8%                            26 28.3%                                   17 18.5%    Sum 261   62 23.7%                     56 21.5%                            70 26.8%                                   73 28.0%    __________________________________________________________________________

Data in Table IV-E-2 showed an expected segregation in F2 calli. F2plants were also used for this evaluation. The F2 plants in a field werepainted with 1% Ignite to verify bar expression and leaf-punches fromall the F2 plants were stained with X-gluc to verify GUS expression. Theresults were reported in Table IV-F-2. The 4 expected phenotypes wereobserved in this F2 population.

For F2 plant segregation:

                                      TABLE IV-F-2    __________________________________________________________________________        Total    F1  F2  GUS+/Ignite+                    GUS+/Ignite+                           GUS+/Ignite-                                   GUS-/Ignite-    Plant        Plants            No. %   No.                       %   No. %   No.                                      %    __________________________________________________________________________    26-2        19  4   21.1%                    4  21.1%                           7   36.8%                                   4  21.1%    26-21        20  4   20.0%                    8  40.0%                           6   30.0%                                   2  10.0%    27-17        20  6   30.0%                    7  35.0%                           4   20.0%                                   3  15.0%    28-19        17  5   29.4%                    4  23.5%                           4   23.5%                                   4  23.5%    32-6        18  7   38.9%                    4  22.2%                           3   16.7%                                   4  22 2%    37-17        19  2   10.5%                    10 52.6%                           3   15.8%                                   4  21.1%    64-10        19  3   15.8%                    6  31.6%                           2   10.5%                                   8  42.1%    64-12        17  2   11.8%                    5  29.4%                           2   11.8%                                   8  47.1%    Sum 149 33  22.1%                    48 32.2%                           31  20.8%                                   37 24.8%    __________________________________________________________________________

The F2 leaf tissues from these 4 classes will be subjected to Southernanalysis to confirm their genotype at the molecular level.

G. FLP-mediated FRT Excision in the Meristem to Produce Large ChimericSectors

In addition to showing FLP-mediated transgene excision in suspensioncells, callus and immature embryo cells (and progenitor cells), we alsodemonstrated efficient excision in meristem tissue to produce heritablesomatic sectors. F3-generation immature embryos from the original event776.89-19-5 were used as starting material. Ears were harvested 9-10days after pollination, when the embryos were at the coleoptilar stageof development. At this point, the apical meristem is exposed for DNAdelivery. A total of 330 coleoptilar-stage embryos were placed ontoMS-based medium with 15% sucrose. After sitting on this medium at 27° C.overnight in the dark, the meristems were bombarded with PHP5096. DNAprecipitation was done using 1.0 um tungsten particles using standardmethods (see Klein et al, ibid). Bombardment was done using a BioradPDS-1000 with the stage set at a distance of 10 cm from the acceleratorstopping screen, using a vacuum of 28 mm Hg. Approximately 1.6 ug DNAwas used per shot. After bombardment, the embryos were placed back ontoMS medium+15% sucrose which promotes embryo maturation. After one week,the embryos were moved onto MS+3% sucrose for germination and moved intolighted growth chambers. As the plants grew, leaf samples were taken andassayed for GUS activity using published methods for GUS histochemicalstaining (Jefferson (1987)). A total of 29 plants had large sectors andwere taken to the greenhouse for further analysis. In 8 of these plants,the sectors appeared to stop (which is consistent with fate developmentstudies), but in 21 plants the sectors persisted into the mature plant.Non-sectored control plants (bombarded without DNA) exhibited theunaltered transgenic phenotype of this event; i.e. all leaves wereBasta-resistant and GUS-negative (no staining). FLP-induced sectors hadlost Basta-resistance and were now sensitive, and had gained positiveGUS expression (blue staining). Nine plants had large, persistentsectors that went all the way into the tassel, and all 9 of thesetransmitted the "excision-phenotype" to their progeny through thechimeric tassel. Six plants in this group also had sectors runningthrough the ear, and transmitted through seed. This represents almost a3% transmission frequency of the bar-excised/GUS-activated locus throughsomatically induced chimeric sectors into progeny.

This result demonstrated that FLP-mediated excision is efficient whenFLP is expressed in the meristem, and this can produce large"excision-sectors" in the resultant plants.

H. Overall Conclusions for Evaluation of Stable Expression in Plants.

From the above data, it can be concluded that FLP recombinase has beensuccessfully used for site-specific recombination in maize. This hasbeen demonstrated in callus, in scutellar cells of immature embryos andin the meristem. Based on these studies, it is expected thatFLP-mediated excision would work in all maize tissues in which FLP isexpressed. The utility of inherited FLP activity and accessibleFRT-flanked loci in the genome have also been demonstrated.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 4    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1272 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (vi) ORIGINAL SOURCE:    #sequence (optimized)M: Synthetic    #ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ    - ATGCCCCAGT TCGACATCCT CTGCAAGACC CCCCCCAAGG TGCTCGTGAG GC - #AGTTCGTG      60    - GAGAGGTTCG AGAGGCCCTC CGGCGAGAAG ATCGCCCTCT GCGCCGCCGA GC - #TCACCTAC     120    - CTCTGCTGGA TGATCACCCA CAACGGCACC GCCATTAAGA GGGCCACCTT CA - #TGTCATAC     180    - AACACCATCA TCTCCAACTC CCTCTCCTTC GACATCGTGA ACAAGTCCCT CC - #AGTTCAAA     240    - TACAAGACCC AGAAGGCCAC CATCCTCGAG GCCTCCCTCA AGAAGCTCAT CC - #CCGCCTGG     300    - GAGTTCACCA TCATCCCCTA CTACGGCCAG AAGCACCAGT CCGACATCAC CG - #ACATCGTG     360    - TCATCCCTCC AGCTTCAGTT CGAGTCCTCC GAGGAGGCTG ACAAGGGCAA CT - #CCCACTCC     420    - AAGAAGATGC TGAAGGCCCT CCTCTCCGAG GGCGAGTCCA TCTGGGAGAT CA - #CCGAGAAG     480    - ATCCTCAACT CCTTCGAGTA CACCTCCAGG TTCACTAAGA CCAAGACCCT CT - #ACCAGTTC     540    - CTCTTCCTCG CCACCTTCAT CAACTGCGGC AGGTTCTCAG ACATCAAGAA CG - #TGGACCCC     600    - AAGTCCTTCA AGCTCGTGCA GAACAAGTAC CTCGGCGTGA TCATCCAGTG CC - #TCGTGACC     660    - GAGACCAAGA CCTCCGTGTC CAGGCACATC TACTTCTTCT CCGCTCGCGG CA - #GGATCGAC     720    - CCCCTCGTGT ACCTCGACGA GTTCCTCAGG AACTCAGAGC CCGTGCTCAA GA - #GGGTGAAC     780    - AGGACCGGCA ACTCCTCCTC CAACAAGCAG GAGTACCAGC TCCTCAAGGA CA - #ACCTCGTG     840    - AGGTCCTACA ACAAGGCCCT CAAGAAGAAC GCCCCCTACT CCATCTTCGC CA - #TCAAGAAC     900    - GGCCCCAAGT CCCACATCGG TAGGCACCTC ATGACCTCCT TCCTCTCAAT GA - #AGGGCCTC     960    - ACCGAGCTCA CCAACGTGGT GGGCAACTGG TCCGACAAGA GGGCCTCCGC CG - #TGGCCAGG    1020    - ACCACCTACA CCCACCAGAT CACCGCCATC CCCGACCACT ACTTCGCCCT CG - #TGTCAAGG    1080    - TACTACGCCT ACGACCCCAT CTCCAAGGAG ATGATCGCCC TCAAGGACGA GA - #CTAACCCC    1140    - ATCGAGGAGT GGCAGCACAT CGAGCAGCTC AAGGGCTCCG CCGAGGGCTC CA - #TCAGGTAC    1200    - CCCGCCTGGA ACGGCATCAT CTCCCAGGAG GTGCTCGACT ACCTCTCCTC CT - #ACATCAAC    1260    #     1272    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 423 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Saccharomyce - #s cerevisiae    #ID NO:2: (xi) SEQUENCE DESCRIPTION: SEQ    -      Met Pro Gln Phe Asp Ile Leu Cys - # Lys Thr Pro Pro Lys Val Leu    Val    #   15    -      Arg Gln Phe Val Glu Arg Phe Glu - # Arg Pro Ser Gly Glu Lys Ile    Ala    #                 30    -      Leu Cys Ala Ala Glu Leu Thr Tyr - # Leu Cys Trp Met Ile Thr His    Asn    #             45    -      Gly Thr Ala Ile Lys Arg Ala Thr - # Phe Met Ser Tyr Asn Thr Ile    Ile    #         60    -      Ser Asn Ser Leu Ser Phe Asp Ile - # Val Asn Lys Ser Leu Gln Phe    Lys    #     80    -      Tyr Lys Thr Gln Lys Ala Thr Ile - # Leu Glu Ala Ser Leu Lys Lys    Leu    #   95    -      Ile Pro Ala Trp Glu Phe Thr Ile - # Ile Pro Tyr Tyr Gly Gln Lys    His    #                110    -      Gln Ser Asp Ile Thr Asp Ile Val - # Ser Ser Leu Gln Leu Gln Phe    Glu    #            125    -      Ser Ser Glu Glu Ala Asp Lys Gly - # Asn Ser His Ser Lys Lys Met    Leu    #        140    -      Lys Ala Leu Leu Ser Glu Gly Glu - # Ser Ile Trp Glu Ile Thr Glu    Lys    #    160    -      Ile Leu Asn Ser Phe Glu Tyr Thr - # Ser Arg Phe Thr Lys Thr Lys    Thr    #   175    -      Leu Tyr Gln Phe Leu Phe Leu Ala - # Thr Phe Ile Asn Cys Gly Arg    Phe    #                190    -      Ser Asp Ile Lys Asn Val Asp Pro - # Lys Ser Phe Lys Leu Val Gln    Asn    #            205    -      Lys Tyr Leu Gly Val Ile Ile Gln - # Cys Leu Val Thr Glu Thr Lys    Thr    #        220    -      Ser Val Ser Arg His Ile Tyr Phe - # Phe Ser Ala Arg Gly Arg Ile    Asp    #    240    -      Pro Leu Val Tyr Leu Asp Glu Phe - # Leu Arg Asn Ser Glu Pro Val    Leu    #   255    -      Lys Arg Val Asn Arg Thr Gly Asn - # Ser Ser Ser Asn Lys Gln Glu    Tyr    #                270    -      Gln Leu Leu Lys Asp Asn Leu Val - # Arg Ser Tyr Asn Lys Ala Leu    Lys    #            285    -      Lys Asn Ala Pro Tyr Ser Ile Phe - # Ala Ile Lys Asn Gly Pro Lys    Ser    #        300    -      His Ile Gly Arg His Leu Met Thr - # Ser Phe Leu Ser Met Lys Gly    Leu    #    320    -      Thr Glu Leu Thr Asn Val Val Gly - # Asn Trp Ser Asp Lys Arg Ala    Ser    #   335    -      Ala Val Ala Arg Thr Thr Tyr Thr - # His Gln Ile Thr Ala Ile Pro    Asp    #                350    -      His Tyr Phe Ala Leu Val Ser Arg - # Tyr Tyr Ala Tyr Asp Pro Ile    Ser    #            365    -      Lys Glu Met Ile Ala Leu Lys Asp - # Glu Thr Asn Pro Ile Glu Glu    Trp    #        380    -      Gln His Ile Glu Gln Leu Lys Gly - # Ser Ala Glu Gly Ser Ile Arg    Tyr    #    400    -      Pro Ala Trp Asn Gly Ile Ile Ser - # Gln Glu Val Leu Asp Tyr Leu    Ser    #   415    -      Ser Tyr Ile Asn Arg Arg Ile                     420    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1272 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Saccharomyce - #s cerevisiae    #ID NO:3: (xi) SEQUENCE DESCRIPTION: SEQ    - ATGCCACAAT TTGGTATATT ATGTAAAACA CCACCTAAGG TGCTTGTTCG TC - #AGTTTGTG      60    - GAAAGGTTTG AAAGACCTTC AGGTGAGAAA ATAGCATTAT GTGCTGCTGA AC - #TAACCTAT     120    - TTATGTTGGA TGATTACACA TAACGGAACA GCAATCAAGA GAGCCACATT CA - #TGAGCTAT     180    - AATACTATCA TAAGCAATTC GCTGAGTTTC GATATTGTCA ATAAATCACT CC - #AGTTTAAA     240    - TACAAGACGC AAAAAGCAAC AATTCTGGAA GCCTCATTAA AGAAATTGAT TC - #CTGCTTGG     300    - GAATTTACAA TTATTCCTTA CTATGGACAA AAACATCAAT CTGATATCAC TG - #ATATTGTA     360    - AGTAGTTTGC AATTACAGTT CGAATCATCG GAAGAAGCAG ATAAGGGAAA TA - #GCCACAGT     420    - AAAAAAATGC TTAAAGCACT TCTAAGTGAG GGTGAAAGCA TCTGGGAGAT CA - #CTGAGAAA     480    - ATACTAAATT CGTTTGAGTA TACTTCGAGA TTTACAAAAA CAAAAACTTT AT - #ACCAATTC     540    - CTCTTCCTAG CTACTTTCAT CAATTGTGGA AGATTCAGCG ATATTAAGAA CG - #TTGATCCG     600    - AAATCATTTA AATTAGTCCA AAATAAGTAT CTGGGAGTAA TAATCCAGTG TT - #TAGTGACA     660    - GAGACAAAGA CAAGCGTTAG TAGGCACATA TACTTCTTTA GCGCAAGGGG TA - #GGATCGAT     720    - CCACTTGTAT ATTTGGATGA ATTTTTGAGG AATTCTGAAC CAGTCCTAAA AC - #GAGTAAAT     780    - AGGACCGGCA ATTCTTCAAG CAATAAACAG GAATACCAAT TATTAAAAGA TA - #ACTTAGTC     840    - AGATCGTACA ATAAAGCTTT GAAGAAAAAT GCGCCTTATT CAATCTTTGC TA - #TAAAAAAT     900    - GGCCCAAAAT CTCACATTGG AAGACATTTG ATGACCTCAT TTCTTTCAAT GA - #AGGGCCTA     960    - ACGGAGTTGA CTAATGTTGT GGGAAATTGG AGCGATAAGC GTGCTTCTGC CG - #TGGCCAGG    1020    - ACAACGTATA CTCATCAGAT AACAGCAATA CCTGATCACT ACTTCGCACT AG - #TTTCTCGG    1080    - TACTATGCAT ATGATCCAAT ATCAAAGGAA ATGATAGCAT TGAAGGATGA GA - #CTAATCCA    1140    - ATTGAGGAGT GGCAGCATAT AGAACAGCTA AAGGGTAGTG CTGAAGGAAG CA - #TACGATAC    1200    - CCCGCATGGA ATGGGATAAT ATCACAGGAG GTACTAGACT ACCTTTCATC CT - #ACATAAAT    1260    #     1272    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 34 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Saccharomyce - #s cerevisiae    #ID NO:4: (xi) SEQUENCE DESCRIPTION: SEQ    #        34        AGAA AGTATAGGAA CTTC    __________________________________________________________________________

We claim:
 1. A nucleotide sequence encoding an FLP recombinase whereinsaid sequence comprises the DNA sequence set forth in SEQ ID NO:
 1. 2. Amethod for excising a target nucleotide sequence in a plant cell, saidmethod comprising introducing a nucleotide sequence modified forenhanced expression in a plant, said modified sequence comprising theDNA sequence set forth in SEQ ID NO: 1, wherein said target sequence isflanked by FRT sites.
 3. The method of claim 2 wherein said plant is adicot.
 4. The method of claim 2 wherein said plant is a monocot.
 5. Themethod of claim 4 wherein said monocot is maize.
 6. A transformed plantcontaining within its genome a nucleotide sequence encoding an FLPrecombinase wherein said sequence comprises the DNA sequence set forthin SEQ ID NO:
 1. 7. The plant of claim 6 wherein said plant is a dicot.8. The plant of claim 6 wherein said plant is a monocot.
 9. The plant ofclaim 8 wherein said monocot is maize.
 10. Seed of the plant of claim 6.11. Seed of the plant of claim
 7. 12. Seed of the plant of claim
 8. 13.The plant of claim 6 wherein said plant further comprises integrated FRTsites in its genome.
 14. Seed of the plant of claim
 13. 15. A plant cellproduced by the method of claim
 2. 16. A plant regenerated from theplant cell of claim
 15. 17. The plant of claim 16 wherein said plant isa maize plant.
 18. Seed of the plant of claim 17.